OsAAA1 Gene in Rice: Overexpression Vector Construction and Genetic Transformation

Song Simin, Zheng Qiming, Li Shijiao, Deng Shiqi, Li Kun, Liu Xinqiong

PDF(1923 KB)
PDF(1923 KB)
Chinese Agricultural Science Bulletin ›› 2020, Vol. 36 ›› Issue (12) : 91-96. DOI: 10.11924/j.issn.1000-6850.casb20191100881

OsAAA1 Gene in Rice: Overexpression Vector Construction and Genetic Transformation

Author information +
History +

Abstract

The objectives are to construct the overexpression vector with Flag tagged OsAAA1, obtain positive transgenic plants and detect expression of OsAAA1, in positive transgenic rice. The sequence of OsAAA1, was amplified by polymerase chain reaction (PCR) using the cDNA template of Nip. The PCR product was cloned into Flag-tagged pU1301; after confirmation of PCR, enzyme digestion and sequencing, recombinant plasmid was transformed to Nip by agrobacterium; when positive transgenic plants were verified by molecular identification, the expression of OsAAA1, was detected by Real-time PCR. The recombinant plasmid of OsAAA1,-pU1301-Flag was successfully constructed; after genetic transformation, thirty-three plants were differentiated; twenty-six transgenic plants were verified to be positive by molecular identification; the Real-time PCR results indicated that the expression of OsAAA1, was up-regulated to different degrees in twenty-three plants. Compared with the Nip, the expression of OsAAA1, was over 30 times in eight positive transgenic plants, in five of which the expression was even over 40 times. The overexpression vector with Flag tagged OsAAA1, was successfully constructed. Genetic transformation results showed that OsAAA1, with Flag-tag could be integrated into the genomic DNA of Nip, which could increase the expression of OsAAA1,. This study provides a basis for discovering functional proteins interacting with OsAAA1, in rice by using the Flag tag.

Key words

rice / OsAAA1 gene / pU1301-Flag vector / genetic transformation / molecular identification

Cite this article

Download Citations
Song Simin , Zheng Qiming , Li Shijiao , Deng Shiqi , Li Kun , Liu Xinqiong. OsAAA1 Gene in Rice: Overexpression Vector Construction and Genetic Transformation. Chinese Agricultural Science Bulletin. 2020, 36(12): 91-96 https://doi.org/10.11924/j.issn.1000-6850.casb20191100881

0 引言

水稻(Oryza sativa)是主要的粮食作物,世界上一半的人口以它为主食[1]。稻瘟病是造成水稻产量减少的最重要的病害之一[2]。而目前对稻瘟病的防治主要采用化学防治,但化学防治存在成本高和环境污染严重等不足。所以,水稻抗病品种的选育是最经济有效的措施[3]。但稻瘟病菌群体结构复杂和易发生变异,导致选育的抗病品种种植3~5年后丧失抗性,因此,新抗病基因的挖掘和选育广谱抗病品种是亟待解决的问题。
AAA-ATPase家族是一个庞大的家族,最早是由Erdmann等提出[4]。目前已发现AAA-ATPase基因家族成员参与许多细胞内生理活动,比如蛋白降解,线粒体DNA复制,转录后调控以及细胞周期调控等等[5,6,7,8]。近几年有关AAA-ATPase基因家族成员的研究较多。在拟南芥中发现AAA-ATPase家族基因参与ABA信号途径、蛋白降解、植物发育和抗旱性等活动[9,10]。烟草中NtAAA1具有对TMV侵染的抗性[11]。在水稻中发现Lrd6-6LMR能增强水稻的免疫[12,13]
OsAAA1属于AAA-ATPase基因家族成员,是本实验室前期通过参与稻瘟病抗性反应的基因芯片筛选获得,同时发现水杨酸与稻瘟病菌均可诱导OsAAA1基因上调表达,并且分析发现其启动子区域可能存在WROXATNPRI顺式作用元件等相关研究[14,15]。通过构建OsAAA1原核表达载体,已成功表达出可溶性的OsAAA1蛋白[16]。同时已构建得到PR1b诱导型启动子OsAAA1转基因植株,发现该基因对稻瘟病具有很好的抗性[17]。本研究拟构建一个带Flag标签的OsAAA1的超表达载体并获得其转基因植株,为挖掘出与OsAAA1潜在的互作蛋白奠定基础,从而深入研究OsAAA1的抗病的分子机理。

1 材料与方法

1.1 供试材料

大肠杆菌DH5α、根癌农杆菌EHA105和日本晴均由中南民族大学生命科学学院提供。pU1301-Flag载体由华中农业大学提供。

1.2 实验试剂

质粒少量提取试剂盒、凝胶回收试剂盒、凝胶清洁试剂盒均购自Axygen公司。限制性内切酶Quick Cut BamHI和Quick Cut KpnI、DNA分子量Marker、Trizol、反转录试剂盒(PrimeScriptTM RT reagent Kit with gDNA Eraser)和rTaq酶均购自Takara公司。2×Phanata Max Master Mix购自南京诺唯赞生物公司。Ligation high购自TOYOBO公司。引物合成由南京金斯瑞生物科技有限公司完成。测序由武汉天一辉远生物公司完成。

1.3 实验方法

1.3.1 OsAAA1-pU1301-Flag载体构建 根据NCBI数据库中OsAAA1的序列号XM_015775639.2的cDNA序列与pU1301-Flag的多克隆位点设计引物,引物序列见表1;然后进行常规PCR扩增,扩增体系:ddH2O 18 µL,2×PhanataMax Master Mix 25 µL,OsAAA1 KpnIF (10 µmol/L) 2 µL,OsAAA1 BamHIR (10 µmol/L) 2 µL,日本晴cDNA 3 µL。PCR反应程序:95℃预变性3 min;95℃变性30 s,62℃退火30 s,72℃延伸1 min 40 s,重复34个循环;72℃延伸7 min;12℃保存。PCR扩增完成后采用1%的琼脂糖凝胶进行电泳回收目的片段。将回收的目的片段进行酶切纯化后与线性空载体进行连接。连接体系为:纯化的PCR目的片段3 µL,线性空载体1 µL,高效连接酶4 µL,16℃连接2 h。连接完成后转化大肠杆菌DH5α感受态细胞中,使用卡那霉素进行筛选培养。初步筛选出的单克隆进行扩大培养,然后提取质粒进行PCR检测与酶切鉴定,同时最后进行测序确认,完成载体构建。测序成功的OsAAA1-PU1301-Flag载体转化EHA105农杆菌感受态细胞,卡那霉素与氯霉素筛选培养的单克隆用于后续水稻遗传转化。
表1 本研究所使用的引物序列
引物名称 序列(5’-3’)
OsAAA1KpnIF CGGGGTACCATGGAGGCGACGTCGTCGTCGTCGT
OsAAA1BamHIR CTTGGATCCCTTATCCTTCCCGACCACTTCTACATC
UbiF TTGTCGATGCTCACCCTGTT
actinF CTCAACCCCAAGGCTAACAG
actinR ACCTCAGGGCATCGGAAC
eEF1aQRTF TTTCACTCTTGGTGTGAAGCAGAT
eEF1aQRTR GACTTCCTTCACGATTTCATCGTAA
OsAAA1QRTF GGCCAAGACATACCTCGACGT
OsAAA1QRTR GCTCTTGGGCGTCAGGTTCT
1.3.2 水稻遗传转化 选取颗粒饱满的日本晴种子进行愈伤诱导;0.9%的次氯酸钠进行种子消毒后采用MS诱导培养基诱导培养10~15天;诱导愈伤后使用农杆菌悬浮液浸染共培养36 h;用无菌水和含有羧苄青霉素与头孢霉素的无菌水清洗愈伤后,将其转移至含有潮霉素的筛选培养基中进行筛选培养30天左右;将长出带有绿点的愈伤转移至含有潮霉素的分化培养基中分化,直至长出小苗;待小苗长至2~3 cm时,将其转移到含抗生素的生根培养基中进行生根10天左右;炼苗3天左右转移至土壤生长,初步获得阳性植株。
1.3.3 转基因植株阳性鉴定 筛选阳性植株分别进行DNA水平检测和RNA水平表达量检测。
采用CTAB法进行转基因植株叶片DNA提取,提取后的DNA进行PCR检测。
DNA提取具体步骤如下:剪取水稻叶片2~3 cm进行液氮研磨至白粉状;每管加入预热的1.67%的CTAB,65℃裂解植物组织1 h,每10 min摇晃混匀;12000 r/min离心10 min,吸取上清于新的EP管中;加入等体积的氯仿异戊醇(24:1)振荡混匀10 min,12000 r/min离心10 min,吸取上清于新的EP管中;加入2/3体积的异丙醇沉淀DNA大约30 min;使用75%的乙醇清洗DNA沉淀2次;自然条件下晾干DNA至透明胶状,根据DNA沉淀多少加入TE溶液溶解DNA,-20℃保存备用。
根据载体序列设计一条正向引物UbiF,与扩增目的序列的反向引物OsAAA1BamHIR配对,进行PCR扩增,产物大小为1700 bp左右。PCR反应体系为:ddH2O 10.625 µL,10×Buffer 1.5 µL,2.5 mmol/µL dNTPs 1.2 µL,rTaq酶0.075 µL,10 mmol/L的正、反引物各0.3 µL,50 ng/µL DNA模板1 µL。PCR扩增程序为:95℃预变性5 min;95℃变性30 s,63℃退火30 s,72℃延伸1 min 40 s,34个重复;72℃延伸7 min;12℃保存。扩增完成后采用1%琼脂糖凝胶进行电泳检测。
采用Trizol法进行转基因植株叶片RNA提取,提取后的RNA使用去除基因组DNA的反转录试剂盒进行反转;反转后的cDNA样品进行质量检测,主要进行常规PCR扩增管家基因actin的片段,引物序列见表1,片段大小为400 bp左右;确定cDNA中无基因组污染后进行荧光定量PCR,选用延伸因子eEF1a作为内参基因,引物序列见表1,检测转基因植株OsAAA1的表达量。

2 结果与分析

2.1 OsAAA1目的片段扩增

以日本晴的cDNA为模板,使用高保真酶进行PCR扩增,扩增后电泳检测结果见图1,产物大小1560 bp。图1中最亮的条带是OsAAA1目的条带。将正确的条带进行切胶回收,回收后的片段进行BamHI与KpnI双酶切,酶切后进行清洁纯化用于后续连接。
图1 OsAAA1目的片段扩增
M:DL2000;1~18:单克隆。

Full size|PPT slide

2.2 OsAAA1-pU1301-Flag阳性克隆鉴定

酶切后的目的片段与线性载体连接后转化DH5α,卡那霉素LB培养基筛选阳性克隆。阳性克隆扩大培养后提取质粒,进行质粒PCR检测与酶切鉴定。质粒PCR采用UbiF与OsAAA1BamHIR引物扩增,片段大小为1700 bp左右,结果见图2。同时采用BamHI与KpnI进行双酶切,酶切后有两条带,一条带大小为14336 bp,另外一条带大小为1560 bp,结果见图3。最后6号克隆测序结果正确,并命名为OsAAA1-PU1301-Flag,载体构建完成。
图2 OsAAA1-pU1301-Flag质粒PCR鉴定
M:DL2000;1~3:Nip的cDNA

Full size|PPT slide

图3 OsAAA1-pU1301-Flag的BamHI与KpnI双酶切鉴定。。
M:DL15000;1~18:单克隆;19:pU1301-Flag空载体

Full size|PPT slide

2.3 水稻遗传转化

将构建完成的OsAAA1-pU1301-Flag载体电激转化EHA105,氯霉素和卡那霉素筛选培养获得阳性克隆。使用农杆菌介导浸染水稻种子愈伤的方法进行遗传转化。首先选取颗粒饱满的日本晴种子进行愈伤组织诱导;然后使用农杆菌悬浮液浸染愈伤组织,进行共培养;共培养完成后在暗培养室进行筛选培养;待筛选长出新的愈伤组织并带有绿点后进行分化;分化出小苗之后进行生根培养;小苗生根后炼苗移至土壤中。各时期的水稻组织形态见图4
图4 农杆菌介导的水稻遗传转化的各时期
A:愈伤组织诱导,B:共培养,C:愈伤组织筛选,D:愈伤组织分化,E:生根培养,F:幼苗移栽

Full size|PPT slide

2.4 转基因植株T0代阳性鉴定

采用CTAB法提取T0代转基因植株的DNA,并利用UbiF与OsAAA1BamHIR引物对其DNA进行分子鉴定,产物大小为1700 bp左右。分子鉴定结果见图5。通过对33株转基因植株进行鉴定,其中只有26株DNA扩增出正确的条带,初步确定这26株为阳性转基因植株。
图5 转基因植株的T0代的分子鉴定
M:DL2000;1~22,25~35:转基因植株;23,36:OsAAA1-pU1301-Flag阳性质粒;24,37:Nip

Full size|PPT slide

2.5 阳性转基因植株的表达量检测

由于幼苗在移栽种植过程中,造成3株阳性植株的死亡,所以后续对剩下的23株转基因植株进行表达量检测。提取23株阳性转基因植株的RNA,提取后反转成cDNA。以管家基因actin的引物对cDNA进行质量检测,扩增出与DNA不一样的单一的特异性条带,条带大小为450 bp,结果见图6。结果证明无基因组DNA的污染,可以用于后续对其进行表达量检测。表达量检测使用eEF1a作为内参基因,对转基因植株的OsAAA1的表达量进行检测分析,检测结果见图7。以受体材料日本晴作为对照,结果显示23株转基因植株的OsAAA1的表达均不同程度上调,其中编号为17的植株上调达到72倍。
图6 阳性转基因植株的cDNA的质量检测
M:DL2000;1~23:阳性转基因植株的cDNA;24:Nip的DNA

Full size|PPT slide

图7 阳性转基因植株的OsAAA1的表达量检测

Full size|PPT slide

3 结论与讨论

长久以来,基因功能研究策略采用功能失活和功能补偿,其中功能补偿常采取超量表达的手段[18]。基因超表达主要是通过超表达载体的构建和转基因手段使目的基因在转化植株中实现超表达来完成的[19]。本研究以日本晴的cDNA为模板,将OsAAA1基因克隆至一个带Flag标签的超表达载体pU1301。Flag标签蛋白为编码8个氨基酸的亲水多肽(DYKDDDDK),是一种促溶解度标签[20]。Flag作为融合表达标签,其通常不会与目的蛋白相互作用并且通常不会影响目的蛋白的功能和性质[21]。成功构建OsAAA1-pU1301-Flag载体并通过遗传转化获得阳性转基因植株,不仅实现OsAAA1基因在日本晴植株中过表达,而且能够简便检测和纯化OsAAA1蛋白。为后续直接在水稻植物体内挖掘出与OsAAA1互作的功能蛋白奠定基础。
pU1301-Flag超表达载体是pU1301载体改造形成的,Flag标签位于BamHI酶切位点后面。由于其位置的特殊性,采用pU1301-Flag载体时,只能选用KpnI与BamHI两个酶切位点,对于能够连接的目的基因具有很大局限性。解决该问题可通过增加酶切位点,但具有一定的繁琐性。
OsAAA1是一个潜在的抗病基因,但目前有关OsAAA1的分子调控机制及抗病机理是未知的。本研究构建了含有Flag标签的OsAAA1的组成型表达载体。采用常见的分子克隆技术进行载体构建;对单克隆进行了质粒PCR检测与酶切检测双重鉴定,确保载体构建的正确性;最后通过测序检测保证基因序列未发生突变,为后续遗传转化奠定基础。水稻遗传转化的各个过程中,正确使用抗生素及其浓度。分化及生根培养基中采用筛选培养基中潮霉素浓度的一半,避免假阳性结果的同时保证转基因植株的存活率。目前通过对转基因植株进行DNA分子检测及基因表达水平检测,转基因植株的阳性率为79%,并且23株阳性转基因植株OsAAA1基因的表达与日本晴相比均有不同程度的上调,同时具有上调达到70倍以上的阳性转基因植株。为后续蛋白表达纯化及蛋白功能研究奠定基础。本研究可提供一种通过基因超表达标签载体研究基因功能的思路。

References

[1]
Khush G S . What it will take to feed 5.0 billion rice consumers in 2030[J]. Plant Molecular Biology, 2005,59(1):1-6.
Major advances have occurred in rice production due to adoption of green revolution technology. Between 1966 and 2000, the population of densely populated low income countries grew by 90% but rice production increased by 130% from 257 million tons in 1966 to 600 million tons in 2000. However, the population of rice consuming countries continues to grow and it is estimated that we will have to produce 40 more rice in 2030. This increased demand will have to be met from less land, with less water, less labor and fewer chemicals. To meet the challenge of producing more rice from suitable lands we need rice varieties with higher yield potential and greater yield stability. Various strategies for increasing the rice yield potential being employed include: (1) conventional hybridization and selection procedures, (2) ideotype breeding, (3) hybrid breeding, (4) wide hybridization and (5) genetic engineering. Various conventional and biotechnology approach are being employed to develop durable resistance to diseases and insect and for tolerance to abiotic stresses. The availability of the rice genome sequence will now permit identification of the function of each of 60,000 rice genes through functional genomics. Once the function of a gene is identified, it will be possible to develop new rice varieties by introduction of the gene through traditional breeding in combination with marker aided selection or direct engineering of genes into rice varieties.
[2]
Skamnioti P, Gurr S J . Against the grain: safeguarding rice from rice blast disease[J]. Trends in Biotechnology, 2009,27(3):141-150.
Rice is the staple diet of more than three billion people. Yields must double over the next 40 years if we are to sustain the nutritional needs of the ever-expanding global population. Between 10% and 30% of the annual rice harvest is lost due to infection by the rice blast fungus Magnaporthe oryzae. Evaluation of genetic and virulence diversity of blast populations with diagnostic markers will aid disease management. We review the M. oryzae species-specific and cultivar-specific avirulence determinants and evaluate efforts towards generating durable and broad-spectrum resistance in single resistant cultivars or mixtures. We consider modern usage of fungicides and plant defence activators, assess the usefulness of biological control and categorize current approaches towards blast-tolerant genetically modified rice.
[3]
Zhang Y X, Yang J Y, Shan Z L , et al. Substitution mapping of QTLs for blast resistance with SSSLs in rice (Oryza sativa L.)[J]. Euphytica, 2012,184(1):141-150.
Rice blast, caused by fungal pathogen Magnaporthe grisea Barr., is one of the most devastating rice diseases worldwide. It has greatly affected the rice production and quality. Development of resistant cultivars is the most effective and economical way for controlling this disease in rice. In this study, 114 of the single-segment substitution lines (SSSLs) in rice were inoculated at seedling stage by 16 rice blast isolates. The substituted segments in the 114 SSSLs distributed on 12 chromosomes with coverage of 57.32% of rice genome. Fifteen of the SSSLs were different in blast resistance from the HJX74 recipient. The SSSL W23-7-6-5-2-2 showed 100% of resistance frequency. A total of 11 QTLs for blast resistance were detected on chromosomes 1, 2, 3, 6, 10, 11 and 12 in rice. They were mapped at chromosomal intervals of 2.2-46.2 cM, of which 6 QTLs were mapped at less than 10.0 cM. Six of the 11 QTLs were first reported in this paper.
[4]
Erdmann R, Wiebel F F, Flessau A , et al. PAS1, a yeast gene required for peroxisome biogenesis, encodes a member of a novel family of putative ATPases[J]. Cell, 1991,64(3):499-510.
PAS genes are required for peroxisome biogenesis in the yeast S. cerevisiae. Here we describe the cloning, sequencing, and characterization of the PAS1 gene. Its gene product, Pas1p, has been identified as a rather hydrophilic 117 kd polypeptide. The predicted Pas1p sequence contains two putative ATP-binding sites and reveals a structural relationship to three other groups of proteins associated with different biological processes such as vesicle-mediated protein transport (NSF and Sec18p), control of cell cycle (Cdc48p, VCP, and p97-ATPase), and modulation of gene expression of the human immunodeficiency virus (TBP-1). The proteins share a highly conserved domain of about 185 amino acids including a consensus sequence for ATP binding. We suggest that these proteins are members of a novel family of putative ATPases and may be descendants of one common ancestor.
[5]
Yedidi R S, Petra W, Cordula E . AAA-ATPases in Protein Degradation[J]. Frontiers in Molecular Biosciences, 2017,4:42-43.
Proteolytic machineries containing multisubunit protease complexes and AAA-ATPases play a key role in protein quality control and the regulation of protein homeostasis. In these protein degradation machineries, the proteolytically active sites are formed by either threonines or serines which are buried inside interior cavities of cylinder-shaped complexes. In eukaryotic cells, the proteasome is the most prominent protease complex harboring AAA-ATPases. To degrade protein substrates, the gates of the axial entry ports of the protease need to be open. Gate opening is accomplished by AAA-ATPases, which form a hexameric ring flanking the entry ports of the protease. Protein substrates with unstructured domains can loop into the entry ports without the assistance of AAA-ATPases. However, folded proteins require the action of AAA-ATPases to unveil an unstructured terminus or domain. Cycles of ATP binding/hydrolysis fuel the unfolding of protein substrates which are gripped by loops lining up the central pore of the AAA-ATPase ring. The AAA-ATPases pull on the unfolded polypeptide chain for translocation into the proteolytic cavity of the protease. Conformational changes within the AAA-ATPase ring and the adjacent protease chamber create a peristaltic movement for substrate degradation. The review focuses on new technologies toward the understanding of the function and structure of AAA-ATPases to achieve substrate recognition, unfolding and translocation into proteasomes in yeast and mammalian cells and into proteasome-equivalent proteases in bacteria and archaea.
[6]
Li P C, Li K, Wang J , et al. The AAA-ATPase MIDASIN 1 Functions in Ribosome Biogenesis and Is Essential for Embryo and Root Development[J]. PLANT PHYSIOLOGY, 2019.
Ribosome biogenesis is an orchestrated process that relies on many assembly factors. The AAA-ATPase Midasin 1 (Mdn1) functions as a ribosome assembly factor in yeast (Saccharomyces cerevisiae), but the roles of MDN1 in Arabidopsis (Arabidopsis thaliana) are poorly understood. Here, we showed that the Arabidopsis null mutant of MDN1 is embryo-lethal. Using the weak mutant mdn1-1, which maintains viability, we found that MDN1 is critical for the regular pattern of auxin maxima in the globular embryo and functions in root meristem maintenance. By detecting the subcellular distribution of ribosome proteins, we noted that mdn1-1 impairs nuclear export of the pre-60S ribosomal particle. The processing of ribosomal precusor RNAs, including 35S, 27SB, and 20S, is also affected in this mutant. MDN1 physically interacts with PESCADILLO2 (PES2), an essential assembly factor of the 60S ribosome, and the observed mislocalization of PES2 in mdn1-1 further implied that MDN1 plays an indispensable role in 60S ribosome biogenesis. Therefore, the observed hypersensitivity of mdn1-1 to a eukaryotic translation inhibitor and high-sugar conditions might be associated with the defect in ribosome biogenesis. Overall, this work establishes a role of Arabidopsis MDN1 in ribosome biogenesis, which agrees with its roles in embryogenesis and root development.
[7]
Hiraishi N, Ishida Y I, Nagahama M . AAA-ATPase NVL2 acts on MTR4-exosome complex to dissociate the nucleolar protein WDR74[J]. Biochemical and Biophysical Research Communications, 2015,467(3):6291-6926.
Nuclear VCP-like 2 (NVL2) is a chaperone-like nucleolar ATPase of the AAA (ATPase associated with diverse cellular activities) family, which exhibits a high level of amino acid sequence similarity with the cytosolic AAA-ATPase VCP/p97. These proteins generally act on macromolecular complexes to stimulate energy-dependent release of their constituents. We previously showed that NVL2 interacts with RNA processing/degradation machinery containing an RNA helicase MTR4/DOB1 and an exonuclease complex, nuclear exosome, and involved in the biogenesis of 60S ribosomal subunits. These observations implicate NVL2 as a remodeling factor for the MTR4-exosome complex during the maturation of pre-ribosomal particles. Here, we used a proteomic screen and identified a WD repeat-containing protein 74 (WDR74) as a factor that specifically dissociates from this complex depending on the ATPase activity of NVL2. WDR74 shows weak amino acid sequence similarity with the yeast ribosome biogenesis protein Nsa1 and is co-localized with NVL2 in the nucleolus. Knockdown of WDR74 decreases 60S ribosome levels. Taken together, our results suggest that WDR74 is a novel regulatory protein of the MTR4-exsosome complex whose interaction is regulated by NVL2 and is involved in ribosome biogenesis.
[8]
Xu X, Ji J, Xu Q , et al. The major-effect QTL CsARN6.1 encodes an AAA-ATPase domain-containing protein that is associated with waterlogging stress tolerance through promoting adventitious root formation[J]. The Plant Journal, 2018,93(5):100-105.
In plants, the formation of hypocotyl-derived adventitious roots (ARs) is an important morphological acclimation to waterlogging stress; however, its genetic basis remains fragmentary. Here, through combined use of bulked segregant analysis-based whole-genome sequencing, SNP haplotyping and fine genetic mapping, we identified a candidate gene for a major-effect QTL, ARN6.1, that was responsible for waterlogging tolerance due to increased AR formation in the cucumber line Zaoer-N. Through multiple lines of evidence, we show that CsARN6.1 is the most possible candidate for ARN6.1 which encodes an AAA ATPase. The increased formation of ARs under waterlogging in Zaoer-N could be attributed to a non-synonymous SNP in the coiled-coil domain region of this gene. CsARN6.1 increases the number of ARs via its ATPase activity. Ectopic expression of CsARN6.1 in Arabidopsis resulted in better rooting ability and lateral root development in transgenic plants. Transgenic cucumber expressing the CsARN6.1Asp allele from Zaoer-N exhibited a significant increase in number of ARs compared with the wild type expressing the allele from Pepino under waterlogging conditions. Taken together, these data support that the AAA ATPase gene CsARN6.1 has an important role in increasing cucumber AR formation and waterlogging tolerance.
[9]
Chung K, Tasaka M . RPT2a, a 26S proteasome AAA-ATPase, is directly involved in Arabidopsis CC-NBS-LRR protein uni-1D-Induced signaling pathways[J]. Plant Cell Physiol, 2011,52:1657-1664.
Arabidopsis semi-dominant uni-1D shows both constitutive defense responses and diverse morphological defects. In particular, uni-1D homozygote (uni-1D) mutants exhibit severe phenotypes including not only highly up-regulated pathogenesis related-1(PR-1) gene expression, but also lethality in the early stage of true leaf formation after germination. The gene responsible for the mutant encodes a coiled-coil-nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR)-type R protein that functions in the recognition of pathogen and the triggering of defense responses. However, the molecular basis of how uni-1D can induce these phenotypes was unknown. In this study, we isolated the regulatory particle triple-ATPase (RPT) subunits 2a and 2b, base components of the 19S regulatory particle in the 26S proteasome, as uni-1D-interacting proteins using yeast two-hybrid screening. Genetic studies showed that crossing with the rpt2a mutant reduces the level of uni-1D-induced PR-1 gene expression and suppresses the lethality of uni-1D, by leading to restoration of lost expression of the WUSCHEL gene, which functions to maintain meristem activity, in the shoot apical mersitem of uni-1D. These results suggest that RPT2a is a major interacting partner of uni-1D/UNI, and that the interaction between uni-1D and RPT2a is responsible for activating both morphology and defense signals.
[10]
Zhang B, Van A O, Thatcher L , et al. The mitochondrial outer membrane AAA ATPase AtOM66 affects cell death and pathogen resistance in Arabidopsis thaliana[J]. Plant Journal for Cell & Molecular Biology, 2014,80(4):709-727.
One of the most stress-responsive genes encoding a mitochondrial protein in Arabidopsis (At3g50930) has been annotated as AtBCS1 (cytochrome bc1 synthase 1), but was previously functionally uncharacterised. Here, we show that the protein encoded by At3g50930 is present as a homo-multimeric protein complex on the outer mitochondrial membrane and lacks the BCS1 domain present in yeast and mammalian BCS1 proteins, with the sequence similarity restricted to the AAA ATPase domain. Thus we propose to re-annotate this protein as AtOM66 (Outer Mitochondrial membrane protein of 66 kDa). While transgenic plants with reduced AtOM66 expression appear to be phenotypically normal, AtOM66 over-expression lines have a distinct phenotype, showing strong leaf curling and reduced starch content. Analysis of mitochondrial protein content demonstrated no detectable changes in mitochondrial respiratory complex protein abundance. Consistent with the stress inducible expression pattern, over-expression lines of AtOM66 are more tolerant to drought stress but undergo stress-induced senescence earlier than wild type. Genome-wide expression analysis revealed a constitutive induction of salicylic acid-related (SA) pathogen defence and cell death genes in over-expression lines. Conversely, expression of SA marker gene PR-1 was reduced in atom66 plants, while jasmonic acid response genes PDF1.2 and VSP2 have increased transcript abundance. In agreement with the expression profile, AtOM66 over-expression plants show increased SA content, accelerated cell death rates and are more tolerant to the biotrophic pathogen Pseudomonas syringae, but more susceptible to the necrotrophic fungus Botrytis cinerea. In conclusion, our results demonstrate a role for AtOM66 in cell death and amplifying SA signalling.
[11]
Sugimoto M, Yamaguchi Y, Nakamura K , et al. A hypersensitive response-induced atpase associated with various cellular activities (AAA) protein from tobacco plants[J]. Plant Molecular Biology, 2004,56(6):973-985.
The hypersensitive response (HR) is one of the most critical defense systems in higher plants. In order to understand its molecular basis, we have screened tobacco genes that are transcriptionally activated during the early stage of the HR by the differential display method. Among six genes initially identified, one was found encoding a 57thinspkDa polypeptide with 497 amino acids not showing significant similarity to any reported proteins except for the AAA domain (ATPase associated with various cellular activities) spanning over 230 amino acids. The bacterially expressed protein exhibited ATP hydrolysis activity, and a green fluorescent protein-fusion protein localized in the cytoplasm of onion epidermis cells. The protein was subsequently designated as NtAAA1 (Nicotiana tabacum AAA1). NtAAA1 transcripts were induced 6thinsph after HR onset not only by TMV but also by incompatiblePsuedomonas syringae, indicating that NtAAA1 is under the control of the N-gene with a common role in pathogen responses. Expression of NtAAA1 was induced by jasmonic acid and ethylene, but not by salicylic acid (SA). It also occurred at a high level in SA-deficient tobacco plants upon TMV infection. When NtAAA1 was silenced by the RNAi method, accumulation of transcripts for PR-1a significantly increased during the HR. Treatments with SA induced higher expression of PR-1a and acidic PR-2 in RNAi transgenic plants than in wild-type counterparts. These results suggest that NtAAA1 mitigates the SA signaling pathway, and therefore that NtAAA1 modulates the pathogen response of the host plants by adjusting the HR to an appropriate level.
[12]
Zhu X, Yin J, Liang S , et al. The multivesicular bodies (MVBs)-localized AAA ATPase LRD6-6 inhibits immunity and cell death likely through regulating MVBs-mediated vesicular trafficking in rice[J]. Plos Genetics, 2016,12(9):45-48.
Previous studies have shown that multivesicular bodies (MVBs)/endosomes-mediated vesicular trafficking may play key roles in plant immunity and cell death. However, the molecular regulation is poorly understood in rice. Here we report the identification and characterization of a MVBs-localized AAA ATPase LRD6-6 in rice. Disruption of LRD6-6 leads to enhanced immunity and cell death in rice. The ATPase activity and homo-dimerization of LRD6-6 is essential for its regulation on plant immunity and cell death. An ATPase inactive mutation (LRD6-6E315Q) leads to dominant-negative inhibition in plants. The LRD6-6 protein co-localizes with the MVBs marker protein RabF1/ARA6 and interacts with ESCRT-III components OsSNF7 and OsVPS2. Further analysis reveals that LRD6-6 is required for MVBs-mediated vesicular trafficking and inhibits the biosynthesis of antimicrobial compounds. Collectively, our study shows that the AAA ATPase LRD6-6 inhibits plant immunity and cell death most likely through modulating MVBs-mediated vesicular trafficking in rice.
[13]
Fekih R, Tamiru M, Kanzaki H , et al. The rice (Oryza sativa L.) LESION MIMIC RESEMBLING, which encodes an AAA-type ATPase, is implicated in defense response[J]. Molecular Genetics and Genomics, 2015,290(2):611-622.
Lesion mimic mutants (LMMs) provide a useful tool to study defense-related programmed cell death (PCD) in plants. Although a number of LMMs have been identified in multiple species, most of the candidate genes are yet to be isolated. Here, we report the identification and characterization of a novel rice (Oryza sativa L.) lesion mimic resembling (lmr) mutant, and cloning of the corresponding LMR gene. The LMR locus was initially delineated to 1.2 Mb region on chromosome 6, which was further narrowed down to 155-kb using insertions/deletions (INDELs) and cleavage amplified polymorphic sequence markers developed in this study. We sequenced the open reading frames predicted within the candidate genomic region, and identified a G-A base substitution causing a premature translation termination in a gene that encodes an ATPase associated with various cellular activities type (AAA-type) protein. RNA interference transgenic lines with reduced LMR transcripts exhibited the lesion mimic phenotype similar to that of lmr plants. Furthermore, expression of the wild-type LMR in the mutant background complemented the lesion phenotype, confirming that the mutation identified in LMR is responsible for the mutant phenotype. The pathogenesis-related (PR) genes PBZ1 and PR1 were induced in lmr, which also showed enhanced resistance to rice blast (Magnaporthe oryzae) and bacterial blight (Xanthomonas oryzae pv. oryzae), suggesting LMR is a negative regulator of cell death in rice. The identification of lmr and cloning of the corresponding LMR gene provide an additional resource for the study of PCD in plants.
[14]
Chen Y H, Dai K, Zhang H , et al. Spectroscopic and molecular docking study on the interaction between salicylic acid and the induced disease-resistant protein OsAAA1 of rice[J]. SpectrochimicaActaPart A: Molecular and Biomolecular Spectroscopy, 2016,10(9):78-80.
The interaction between salicylic acid (SA) and the induced disease-resistant protein OsAAA1 in rice was studied using spectroscopy and molecular docking. Ultraviolet (UV) absorption spectroscopy demonstrated an interaction between OsAAA1 protein and SA. Spectroscopy showed that this interaction was a dynamic quenching process. Synchronous fluorescence spectroscopy (SFS) further revealed that this interaction caused changes in the microenvironment of tyrosine and tryptophan and that the interaction site was closer to the tryptophan residue. The structural model of protein OsAAA1 was determined by homology modeling method, and the molecular docking simulation diagram of OsAAA1 with SA was obtained. These models, in combination with a Ramachandran plot analysis, showed amino acid residues ranging from position 240 to position 420 as the possible site interacting with SA. Among them, Gly389, Lys257 and Glu425 might be three key amino acids that can form hydrogen bonds with SA.
[15]
邓霄霄, 刘早利, 刘新琼 , 等. 水稻OsAAA1基因启动子的分离与克隆[J]. 分子植物育种, 2017,15(08):2907-2911.
[16]
刘新琼, 徐玮玉, 刘早利 , 等. 水稻OsAAA1蛋白的原核表达载体构建及其可溶性表达研究[J]. 中南民族大学学报:自然科学版, 2015,34(02):18-22.
[17]
张旺, 陈佳莹, 刘新琼 , 等. 水稻诱导抗病基因OsAAA1启动子构建体的遗传转化与诱导表达[J]. 分子植物育种, 2018,16(21):7021-7026.
[18]
杨诗怡, 周小利, 陈志芸 , 等. 植物基因功能验证技术概述[J]. 安徽农业科学, 2017,45(34):136-140.
[19]
黄越敏 . 水稻逆境相关基因的遗传转化和功能鉴定[D]. 武汉:华中农业大学, 2006.
[20]
李忠信 . FLAG标签单克隆抗体的制备、鉴定与应用研究[D]. 郑州:河南工业大学, 2010.
[21]
邹珊珊, 王萍, 魏威 , 等. 携带GFP和FLAG标签的真核表达载体介导细胞珠蛋白在细胞内表达的定位研究[J]. 热带医学杂志, 2014,14(04):456-460,548.

RIGHTS & PERMISSIONS

Copyright reserved © 2020. Chinese Agricultural Association. All articles published represent the opinions of the authors, and do not reflect the official policy of the Chinese Agricultural Association or the Editorial Board, unless this is clearly specified.
Share on Mendeley
PDF(1923 KB)

152

Accesses

0

Citation

Detail

Sections
Recommended

/