TaHPPR Gene in Wheat: Cloning and Expression Analysis

Hao Xiaocong, Wang Weiwei, Zhang Fengting, Sun Rui, Fang Zhaofeng, Liu Shan, Cao Zhishen, Zhu Wengen, Zhao Changping, Wang Dezhou, Tang Yimiao

PDF(1458 KB)
PDF(1458 KB)
Chinese Agricultural Science Bulletin ›› 2021, Vol. 37 ›› Issue (3) : 129-138. DOI: 10.11924/j.issn.1000-6850.casb2020-0010

TaHPPR Gene in Wheat: Cloning and Expression Analysis

Author information +
History +

Abstract

To identify the role of hydroxyphenylpyruvate reductase (HPPR) in wheat stress, this study used homologous cloning to obtain a wheat HPPR gene which named TaHPPR. At the same time, the expressions of HPPR gene in tissues and under stress were analyzed. ‘Taiyuan 806’, ‘Xiaobaimai’, ‘Jingdong 8’ and ‘Jing 411’ were used as materials. We performed RT-qPCR analysis to determine TaHPPR gene expression levels in tissue and under stress. Sequence analysis showed that the TaHPPR gene contained a complete open reading frame of 975 bp, and encoded 324 amino acids. TaHPPR protein contained a NAD-binding domain structure. In addition, phylogenetic analysis indicated that TaHPPR gene was in the same branch of Triticum dicoccoides and their genetic relationships were extremely familiar. Expression profiling revealed that TaHPPR expressed in roots, spikelets (excluding stamens), and leaf sheaths, of which the expression level in roots was the highest. The expression of TaHPPR gene decreased under cold, drought, high salt and ABA stress treatment, In the high-salt resistance variety ‘Jingdong 8’, TaHPPR gene expression increased, while it was suppressed in the sensitive variety ‘Xiaobaimai’ and the moderate sensitive variety ‘Jing411’. The results of subcellular localization show that TaHPPR protein is mainly expressed in mitochondria, and the overexpression of TaHPPR gene may increase the salt tolerance in wheat. This study clarifies the expression characteristics of TaHPPR under adversity stress response and salt stress treatment in different varieties, and provides new gene resources and new ideas for studying the molecular mechanism of wheat resistance breeding.

Key words

wheat / hydroxyphenylpyruvate reductase / abiotic stress / salt resistance / subcellular localization

Cite this article

Download Citations
Hao Xiaocong , Wang Weiwei , Zhang Fengting , Sun Rui , Fang Zhaofeng , Liu Shan , Cao Zhishen , Zhu Wengen , Zhao Changping , Wang Dezhou , Tang Yimiao. TaHPPR Gene in Wheat: Cloning and Expression Analysis. Chinese Agricultural Science Bulletin. 2021, 37(3): 129-138 https://doi.org/10.11924/j.issn.1000-6850.casb2020-0010

0 引言

油甘(Phyllanthus emblica)又称余甘子,是大戟科叶下珠属一类小乔木,主要种植在马来西亚、泰国和印度等热带与亚热带国家,中国云南、广东和福建等南方地区也有广泛分布。油甘是药食兼用水果[1-4],风味独特,营养丰富,尤其维生素C含量远高于其他水果,且含有大量蛋白质和微量元素等,具有消炎、抗氧化、抗癌和抗衰等功效[5-9],受到人们广泛关注。油甘果实可通过腌制、提取、榨汁、发酵等不同方式加工成各种产品[10-11]。随着鲜食果茶的盛行,油甘需求量显著增加,栽培面积也逐年攀升,已成为乡村振兴的重要树种。
油甘果实采后容易被青霉、链格孢属真菌等有害微生物侵染[12-13],同时面临着果实失水率和腐烂率高、褐化快等问题[14],因此油甘果实的采后保鲜是产业化面临的主要问题之一。留树保鲜是通过喷施生长调节剂、覆膜和套袋等方式延长果实采收期,从而错开上市高峰,提升果农收益。早在20世纪80年代,国内外众多学者就相继开展了水果留树保鲜的相关研究,Jacqueline等[15]研究发现留树保鲜不利于早熟葡萄柚的粒化发育;Mnhamed等[16]研究表明2,4-D与GA3混用可延长柑橘留树保鲜的时间和采收期;王春燕等[17]研究红毛丹留树保鲜时发现,40天时红毛丹可溶性固形物和糖的含量显著上升,明确了红毛丹留树保鲜的最佳时间;刘珞忆等[18]研究表明脐橙留树保鲜30天和75天为最佳采收时期。但对油甘留树保鲜的研究鲜见报道,以往研究多集中在采后储藏[19-21]、不同品种成分提取和分析[22-29]、品种选育[30-33]等方面,导致留树保鲜技术在油甘生产中的应用和推广受限。
果实挂果期是决定留树保鲜时长的重要因素,不同水果挂果时间差异较大,如晚熟龙眼最长挂果期仅19天[34],而脐橙的挂果期可达140天[35]。赖多等[36]通过分析10月、12月和次年1月‘白玉油甘’的果实品质,发现留树保鲜3个月后油甘果实单宁含量显著降低,而维生素C含量显著上升。涩味和化渣率是影响油甘鲜食品质的重要因素,油甘在生产中挂果期长达半年之久,但半年后油甘的品质如何变化,如口感、风味和营养价值等升高亦或降低,这方面的研究却未见报道。鉴于此,笔者以晚熟新品种‘上湖仙’油甘[33]为试材,分别在11月、次年1和4月分别采样,测定其果实品质及功能性成分等变化,评价油甘留树保鲜不同时间后的品质优劣,以期为油甘留树保鲜技术的推广应用提供理论指导。

1 材料与方法

1.1 试验材料

‘上湖仙’油甘种植于广东省潮州市果树研究所(23.69°N,116.88°E)。以11月上旬成熟的油甘作对照,留树保鲜果实分别于次年的1月和4月采摘,中间相隔3个月,采样时随机选取大小一致、成熟度相同且无病虫为害的健康油甘果实100个。

1.2 试验方法

‘上湖仙’油甘正季果实(11月上旬成熟)和留树保鲜果实采摘后立即运回广东省农业科学院果树研究所资源与环境研究室处理,经冷冻干燥后粉碎过筛,粉末装于密封离心管中,4℃低温保存待用。采用微量法测定样品中的氨基酸、糖、营养成分、功能性成分和酶活性共24个指标,按试剂盒说明书进行操作,试剂盒均购自苏州科铭生物技术有限公司。

1.3 数据处理与分析

采用Microsoft Excel 2016软件对数据进行处理。采用SPSS 17.0软件进行单因素方差和皮尔森相关性分析。

2 结果与分析

2.1 留树保鲜对油甘氨基酸含量的影响

表1显示,留树保鲜的油甘果实中氨基酸含量与正季采收(11月上旬)相比呈上升趋势,且留树保鲜时间越长氨基酸含量越高。所测氨基酸结果中,除赖氨酸含量在次年1月采收与11月正季采收相比差异不显著外(P>0.05),半胱氨酸、谷氨酸、脯氨酸和羟脯氨酸的含量在留树保鲜3个月和6个月后均显著(P<0.05)上升趋势。其中,半胱氨酸、脯氨酸的含量在11月采收时分别为12.54、0.21 mg/g,留树保鲜至次年1月其含量为17.32、0.37 mg/g,4月采收的含量达到24.14、0.51 mg/g,约为正季采收果实的2倍;谷氨酸、赖氨酸和羟脯氨酸的含量在留树保鲜6个月后的含量分别是正季果的1.23、1.58和1.56倍。可见,‘上湖仙’油甘的氨基酸含量随留树保鲜时间的延长而增加。
表1 留树保鲜对油甘氨基酸的影响 mg/g
氨基酸 11月 1月 4月
半胱氨酸/(mg/g) 12.54±2.93a 17.32±1.88b 24.14±1.04c
谷氨酸/(mg/g) 1.66±0.01a 1.91±0.23b 2.05±0.28c
赖氨酸/(mg/g) 7.87±0.08a 8.14±0.16a 12.40±0.95b
脯氨酸/(mg/g) 0.21±0.01a 0.37±0.02b 0.51±0.01c
羟脯氨酸/(μg/g) 66.64±6.90a 89.31±4.87b 104.05±18.24c
注:同行数据后小写英文字母不同表示差异显著,P<0.05,下同。

2.2 留树保鲜对油甘糖含量的影响

与正季采收相比,‘上湖仙’油甘经留树保鲜后果实中总糖、蔗糖和还原糖的含量均显著(P<0.05)下降趋势,留树保鲜时间越长,下降越显著(表2)。总糖含量从119.16 mg/g(11月采收)下降至98.35 mg/g(1月采收)和78.34 mg/g(4月采收);还原糖含量下降最显著,次年4月采收与11月相比下降了46.9%。可见,留树保鲜不利于总糖、蔗糖和还原糖的积累。
表2 留树保鲜对油甘糖类的影响 mg/g
糖类 11月 1月 4月
总糖 119.16±4.40a 98.35±6.47b 78.34±3.97c
蔗糖 42.34±1.83a 37.61±2.94b 33.59±4.52c
还原糖 58.67±4.81a 44.85±3.76b 31.12±0.88c

2.3 留树保鲜对油甘营养成分的影响

正季油甘果实的水溶性果胶含量为1.87 mg/g,次年1、4月采收的含量分别为1.86、1.81 mg/g,留树保鲜后的含量与正季果相比差异不显著(P>0.05)(表3)。
表3 留树保鲜对油甘营养成分的影响
营养成分 11月 1月 4月
水溶性果胶 1.87±0.18a 1.86±0.18a 1.81±0.05a
还原型抗坏血酸 6.05±0.18a 7.80±0.11b 7.43±0.23b
单宁 5.08±0.07a 3.92±0.26b 3.33±0.48b
纤维素 95.28±8.45a 80.41±2.99b 78.02±3.38b
留树保鲜后的油甘果实中单宁和纤维素含量与正季油甘相比显著降低(P<0.05),还原型抗坏血酸(维生素C)的含量却显著升高,但留树保鲜3个月与6个月相比差异不显著。可见,‘上湖仙’油甘经留树保鲜处理后对果实水溶性果胶的影响不大,但可显著提升还原型抗坏血酸的含量,降低单宁和纤维素的含量。

2.4 留树保鲜对油甘功能性成分的影响

表4数据显示,留树保鲜后油甘的总抗氧化能力、原花青素、类黄酮和总酚含量均呈显著下降趋势(P< 0.05)。油甘果实的总抗氧化能力经留树保鲜3个月和6个月后,分别降低了53.54%和68.75%;原花青素含量降低了46.03%和49.87%;类黄酮含量下降了48.96%和72.73%;总酚含量下降了55.93%和77.75%。尽管‘上湖仙’油甘的留树保鲜不利于原花青素、类黄酮和总酚积累,但这些酚类物质含量的降低有助于减轻果实涩味、提升口感。
表4 留树保鲜对油甘功能性成分的影响
功能性成分 11月 1月 4月
总抗氧化能力/(μmol/mg) 5.92±0.37a 2.75±0.22b 1.85±0.05c
原花青素/(mg/g) 7.80±0.11a 4.21±0.09b 3.91±0.15b
类黄酮/(mg/g) 34.21±1.84a 17.46±0.97b 9.33±0.48c
总酚/(mg/g) 58.02±3.38a 25.57±0.75b 12.91±0.07c

2.5 留树保鲜对油甘酶活性的影响

油甘果实中的8种酶活性在次年1月采收与正季11月采收相比均发生显著变化,其中酸性磷酸酶、过氧化氢酶、多酚氧化酶和乙酰胆碱酯酶含量均显著降低,而碱性磷酸酶、羧酸酯酶、超氧化物歧化酶和过氧化物酶含量显著升高。油甘果实留树保鲜至4月采收时,羧酸酯酶、酸性磷酸酶和多酚氧化酶含量与1月采收的油甘相比差异不显著,即这3种酶活力的变化受留树保鲜时间影响不大;而其他5种酶与1月采收的油甘相比均差异显著(表5)。其中,留树保鲜油甘中4月采收与正季11月采收相比,上升最显著的酶为过氧化物酶,提升了50%,而下降最显著的为酸性磷酸酶,下降了75.42%。综上,油甘的留树保鲜可促进碱性磷酸酶、羧酸酯酶、超氧化物歧化酶和过氧化物酶的积累,同时促使酸性磷酸酶、过氧化氢酶、多酚氧化酶和乙酰胆碱酯酶的降解。
表5 留树保鲜对油甘酶活性的影响
酶活性 11月 1月 4月
碱性磷酸酶/[μmol/(min·g)] 50.64±1.58a 61.51±1.09b 72.73±3.06c
羧酸酯酶/(U/g) 77.62±6.39a 89.43±4.21b 91.06±9.38b
超氧化物歧化酶/(U/g) 166.40±21.27a 180.56±11.09b 186.72±9.23c
过氧化物酶/(U/g) 1020.44±91.48a 1333.57±72.64b 1539.67±86.22c
多酚氧化酶/(U/g) 27.68±4.12a 20.79±3.01b 19.08±2.63b
酸性磷酸酶/[μmol/(min·g)] 4.72±0.01a 1.78±0.03b 1.16±0.04b
过氧化氢酶/[nmol/(min·g)] 291.15±26.22a 202.28±18.49b 162.35±18.23c
乙酰胆碱酯/[nmol/(min·g)] 20.58±4.36a 11.37±3.27b 7.35±2.14c

3 结论

研究结果表明,与正季采收相比,‘上湖仙’油甘留树保鲜后,半胱氨酸、谷氨酸、赖氨酸、脯氨酸和羟脯氨酸及还原型抗坏血酸(维生素C)的含量均显著升高,而糖、原花青素、类黄酮、总酚、单宁和纤维素的含量显著降低。其中,酚类、单宁和纤维素含量降低有利于减轻果实涩味,增加化渣程度;酶活力测定结果显示,酸性磷酸酶、过氧化氢酶、多酚氧化酶和乙酰胆碱酯酶含量均显著降低,而碱性磷酸酶、羧酸酯酶、超氧化物歧化酶和过氧化物酶含量显著升高。综上所述,‘上湖仙’油甘留树保鲜会使抗氧化能力小幅度下降,但可提升油甘的品质和口感,具有良好的商品性,且留树保鲜时间与氨基酸的提升及单宁和纤维素的降低呈正相关,故油甘的最佳留树保鲜时间应为1月前后采摘,因该时间为春节前后,经济效益最高,该方法可在油甘生产中推广应用。

4 讨论

油甘是极具岭南特色的药食兼用型水果,可通过腌制、榨汁和发酵等方式将其加工成果脯、果汁和果酒等商业产品[10-11],油甘的深加工不仅延伸了产业链,也延长了油甘的储藏期。但油甘的深加工往往投入较大,且不利于营养品质保持。近年来,鲜食油甘越来越受到消费者的青睐,但油甘的采后保鲜多以低温储藏为主,低温储藏不仅操作步骤相对复杂,还容易对果实造成一定损耗,导致果实品质降低[19]。留树保鲜作为一种绿色保鲜方式,不仅减少了储藏期间果实营养的损耗,还降低了果实的储藏成本,提高果农收益[36]。‘上湖仙’油甘是笔者及其团队选育出来的晚熟新品种,该果实个大、涩味轻、口感好,适合鲜食,是研究油甘保鲜的良好材料。
本研究发现,‘上湖仙’油甘留树保鲜后总糖含量呈显著下降趋势,这与赖多等[36]研究的‘白玉’油甘及王春燕等[17]对红毛丹留树保鲜的研究结果一致。果实在留树保鲜过程中需要消耗大量营养物质才能维持自身正常的生命活动,随挂果时间延长,消耗量增大,若果园的水肥管理欠缺,树体则不能获取足够的营养物质,最终导致糖含量降低[37-38]。此外,本研究还发现,油甘的留树保鲜一方面可促进氨基酸和还原型抗坏血酸(维生素C)的积累;另一方面可降低单宁、原花青素、类黄酮和总酚和纤维素含量,且增加和下降的趋势均与留树保鲜时间呈正比。单宁、酚类物质以及纤维素与油甘的涩味和化渣程度密切相关,是影响油甘鲜食口感的重要因素之一。油甘留树保鲜至次年采摘会经过低温或霜冻,而环境胁迫可促使蛋白质、氨基酸和维生素的累积[39],同时可降低单宁、酚类物质和纤维素的含量,从而使涩味变轻、化渣变好,口感和营养价值提升。
油甘留树保鲜后的酶活力测定结果表明,过氧化物酶含量显著增加,而多酚氧化酶的含量显著降低。多酚氧化酶是水果褐变的关键酶,该酶活力增加可降低果实品质[40]。研究表明,抗坏血酸可抑制油甘果实中多酚氧化酶的活性,从而延缓油甘褐化速度[41-42]。本研究检测到留树保鲜后的油甘果实中还原型抗坏血酸的含量与正季采摘的油甘相比显著提升,这可能是导致留树保鲜油甘中多酚氧化酶活性降低的主要原因。过氧化物酶是植物细胞壁形成的关键酶,可使细胞壁硬化,从而提高植物的抗逆性[43]。植物细胞壁是抵御外界有害生物及病原微生物的第一道屏障[44],果胶作为构成植物细胞壁的重要成分可被水解为可溶性果胶,在果实成熟过程中可溶性果胶含量逐渐升高[45],促使果实表皮软化,抗病性变差。在本研究中,油甘留树保鲜后的可溶性果胶含量并无显著变化,说明油甘留树保鲜对果实细胞壁的降解影响不大。以上研究结果充分说明,油甘留树保鲜后可延缓其褐变,同时促进新细胞壁的合成,提升果品抗逆性。这也就更好地解释了‘上湖仙’油甘留树保鲜至次年1月或4月时,果品的商品性无显著变化的原因。其他采后保鲜技术虽然可以有效杀灭或抑制微生物活动,干扰果实的呼吸代谢,延缓果实的衰老,但是成本较高、消耗人力,且容易产生病害;而留树保鲜可在不使用任何栽培措施和药剂处理的情况下,改善果实的品质,减少储藏成本、节约人力。生产中油甘的集中上市时间为9月上旬—10月下旬,油甘留树保鲜后良好商品性是提升市场竞争力的前提条件,特别在春节期间上市,普遍售价较高,果农收益大幅提升,但如何更好地提升留树保鲜后油甘的果实品质是下一步研究需要攻克的难题,且后续需针对油甘的配套栽培技术进行研究。

References

[1]
Grant G A. A new family of 2-hydroxyacid dehydrogenases[J]. Biochemical and Biophysical Research Communications, 1990,165(3):1371-1374.
The NADH-dependent hydroxypyruvate reductase from cucumber and the pdxB gene product of E. coli display significant homology to E. coli D-3-phosphoglycerate dehydrogenase. In contrast, these proteins do not display much similarity with other oxidoreductases or with other 2-hydroxyacid dehydrogenases in particular. On the basis of their relatedness and the structure of their substrates, these three enzymes constitute a new family of 2-hydroxyacid dehydrogenases distinct from malate and lactate dehydrogenase.
[2]
Bridge A, Barr R, Morré D J. The plasma membrane NADH oxidase of soybean has vitamin K1 hydroquinone oxidase activity[J]. Biochimica et Biophysica Acta, 2000,1463(2):450-458.
[3]
Häusler E, Petersen M, Alfermann A W. Isolation of protoplasts and vacuoles from cell suspension cultures of Coleus blumei Benth[J]. Plant Cell Reports, 1993,12(9):510-512.
In order to study the accumulation and transport of rosmarinic acid in suspension cells of Coleus blumei we established an efficient method to isolate protoplasts and vacuoles. Protoplasts were disrupted by an osmotic shock in a medium with basic pH containing ethylenediamine tetraacetic acid. The resulting vacuoles were purified on a two-step Ficoll gradient. The comparison of the rosmarinic acid contents of cells, protoplasts and vacuoles showed that the depside is localized in the vacuole. Data concerning the yield and purity of the vacuoles are presented. In addition we show that at the physiological pH of the cytoplasm rosmarinic acid is present almost exclusively as an anion and cannot pass a membrane by simple diffusion. We therefore propose a carrier system for the transport of rosmarinic acid into the vacuole.
[4]
Amesz J. The function of plastoquinone in photosynthetic electron transport[J]. Biochimica et Biophysica Acta (BBA)-Reviews on Bioenergetics, 1973,301:35-51.
[5]
Susan R. Norris, Terrence R. Barrette, Dean Della Penna. Genetic Dissection of Carotenoid Synjournal in Arabidopsis Defines Plastoquinone as an Essential Component of Phytoene Desaturation[J]. The Plant Cell, 1995,7(12):2139-2149.
Carotenoids are C40 tetraterpenoids synthesized by nuclear-encoded multienzyme complexes located in the plastids of higher plants. To understand further the components and mechanisms involved in carotenoid synthesis, we screened Arabidopsis for mutations that disrupt this pathway and cause accumulation of biosynthetic intermediates. Here, we report the identification and characterization of two nonallelic albino mutations, pds1 and pds2 (for phytoene desaturation), that are disrupted in phytoene desaturation and as a result accumulate phytoene, the first C40 compound of the pathway. Surprisingly, neither mutation maps to the locus encoding the phytoene desaturase enzyme, indicating that the products of at least three loci are required for phytoene desaturation in higher plants. Because phytoene desaturase catalyzes an oxidation reaction, it has been suggested that components of an electron transport chain may be involved in this reaction. Analysis of pds1 and pds2 shows that both mutants are plastoquinone and tocopherol deficient, in addition to their inability to desaturate phytoene. Separate steps of the plastoquinone/tocopherol biosynthetic pathway are affected by these two mutations. The pds1 mutation affects the enzyme 4-hydroxyphenylpyruvate dioxygenase because it can be rescued by growth on the product but not the substrate of this enzyme, homogentisic acid and 4-hydroxyphenylpyruvate, respectively. The pds2 mutation most likely affects the prenyl/phytyl transferase enzyme of this pathway. Because tocopherol-deficient mutants in the green alga Scenedesmus obliquus can synthesize carotenoids, our findings demonstrate conclusively that plastoquinone is an essential component in carotenoid synthesis. We propose a model for carotenoid synthesis in photosynthetic tissue whereby plastoquinone acts as an intermediate electron carrier between carotenoid desaturases and the photosynthetic electron transport chain.
[6]
Munné-Bosch, Sergi, Alegre L. The Function of Tocopherols and Tocotrienols in Plants[J]. Critical Reviews in Plant Sciences, 2002,21(1):31-57.
[7]
Xu J J, Fang X, Li C Y, et al. Characterization of Arabidopsis thaliana Hydroxyphenylpyruvate Reductases in the Tyrosine Conversion Pathway[J]. Frontiers in Plant Science, 2018,9:1305.
Tyrosine serves as a precursor to several types of plant natural products of medicinal or nutritional interests. Hydroxyphenylpyruvate reductase (HPPR), which catalyzes the reduction of 4-hydroxyphenylpyruvic acid (pHPP) to 4-hydroxyphenyllactic acid (pHPL), has been shown to be the key enzyme in the biosynthesis of rosmarinic acid (RA) from tyrosine and, so far, HPPR activity has been reported only from the RA-accumulating plants. Here, we show that HPPR homologs are widely distributed in land plants. In Arabidopsis thaliana, which does not accumulate RA at detectable level, two homologs (HPPR2 and HPPR3) are functional in reducing pHPP. Phylogenetic analysis placed HPPR2 and HPPR3 in two separate groups within the HPPR clade, and HPPR2 and HPPR3 are distinct from HPR1, a peroxisomal hydroxypyruvate reductase (HPR). In vitro characterization of the recombinant proteins revealed that HPPR2 has both HPR and HPPR activities, whereas HPPR3 has a strong preference for pHPP, and both enzymes are localized in the cytosol. Arabidopsis mutants defective in either HPPR2 or HPPR3 contained lower amounts of pHPL and were impaired in conversion of tyrosine to pHPL. Furthermore, a loss-of-function mutation in tyrosine aminotransferase (TAT) also reduced the pHPL accumulation in plants. Our data demonstrate that in Arabidopsis HPPR2 and HPPR3, together with TAT1, constitute to a probably conserved biosynthetic pathway from tyrosine to pHPL, from which some specialized metabolites, such as RA, can be generated in specific groups of plants. Our finding may have broad implications for the origins of tyrosine-derived specialized metabolites in general.
[8]
Timm S, Nunes-nesi A, Parnik T, et al. A Cytosolic Pathway for the Conversion of Hydroxypyruvate to Glycerate during Photorespiration in Arabidopsis[J]. The Plant Cell Online, 2008,20(10):2848-2859.
[9]
Timm S, Florian A, Jahnke K, et al. The Hydroxypyruvate-Reducing System in Arabidopsis: Multiple Enzymes for the Same End[J]. Plant Physiology, 2011,155(2):694-705.
[10]
Yuan Ma, Jungen Kang, Jian Wu, et al. Identification of tapetum-specific genes by comparing global gene expression of four different male sterile lines in Brassica oleracea[J]. Plant Molecular Biology, 2015,87(6):541-554.
The tapetum plays an important role in anther development by providing necessary enzymes and nutrients for pollen development. However, it is difficult to identify tapetum-specific genes on a large-scale because of the difficulty of separating tapetum cells from other anther tissues. Here, we reported the identification of tapetum-specific genes by comparing the gene expression patterns of four male sterile (MS) lines of Brassica oleracea. The abortive phenotypes of the four MS lines revealed different defects in tapetum and pollen development but normal anther wall development when observed by transmission electron microscopy. These tapetum displayed continuous defective characteristics throughout the anther developmental stages. The transcriptome from flower buds, covering all anther developmental stages, was analyzed and bioinformatics analyses exploring tapetum development-related genes were performed. We identified 1,005 genes differentially expressed in at least one of the MS lines and 104 were non-pollen expressed genes (NPGs). Most of the identified NPGs were tapetum-specific genes considering that anther walls were normally developed in all four MS lines. Among the 104 NPGs, 22 genes were previously reported as being involved in tapetum development. We further separated the expressed NPGs into different developmental stages based on the MS defects. The data obtained in this study are not only informative for research on tapetum development in B. oleracea, but are also useful for genetic pathway research in other related species.
[11]
Wang G Q, Chen J F, Yi B, et al. HPPR encodes the hydroxyphenylpyruvate reductase required for the biosynjournal of hydrophilic phenolic acids in Salvia miltiorrhiza[J]. Chinese Journal of Natural Medicines, 2017,15(12):45-55.
[12]
Ru M, Wang K, Bai Z, et al. A tyrosine aminotransferase involved in rosmarinic acid biosynjournal in Prunella vulgaris L.[J]. entific Reports, 2017,7(1):4892.
[13]
郭宇, 郝磊, 吕晓玲, 等. 紫苏HPPR基因启动子的克隆及植物表达载体构建[J]. 分子植物育种, 2016,14(2):382-388.
[14]
Tardif G, Kane N A, Adam H, et al. Interaction network of proteins associated with abiotic stress response and development in wheat[J]. Plant Molecular Biology, 2007,63(5):703-718.
Wheat is the most widely adapted crop to abiotic stresses and considered an excellent system to study stress tolerance in spite of its genetic complexity. Recent studies indicated that several hundred genes are either up- or down-regulated in response to stress treatment. To elucidate the function of some of these genes, an interactome of proteins associated with abiotic stress response and development in wheat was generated using the yeast two-hybrid GAL4 system and specific protein interaction assays. The interactome is comprised of 73 proteins, generating 97 interactions pairs. Twenty-one interactions were confirmed by bimolecular fluorescent complementation in Nicotiana benthamiana. A confidence-scoring system was elaborated to evaluate the significance of the interactions. The main feature of this interactome is that almost all bait proteins along with their interactors were interconnected, creating a spider web-like structure. The interactome revealed also the presence of a
[15]
Dan L, Ang L, Xinguo M, et al. Cloning and Characterization of TaPP2AbB\"-α, a Member of the PP2A Regulatory Subunit in Wheat [J]. Plos One, 2014,9(4):e94430-.
[16]
Xu Z S, Chen M, Li L C, et al. Functions of the ERF transcription factor family in plants[J]. Botany-botanique, 2008,86(9):969-977.
[17]
Xu Z S, Chen M, Li L C, et al. Functions and Application of the AP2/ERF Transcription Factor Family in Crop Improvement[J]. Journal of Integrative Plant Biology, 2011(7):64-79.
[18]
李龙, 毛新国, 王景一, 等. 小麦种质资源抗旱性鉴定评价[J]. 作物学报, 2018,044(7):988-999.
[19]
洪琳, 徐磊, 马锦绣, 等. 普通小麦TaTPR1基因的克隆及表达分析[J]. 麦类作物学报, 2014,34(9):1161-1169.
[20]
李楠, 郑勇奇, 丁红梅. 低温胁迫下短枝木麻黄耐寒相关基因的差异表达分析[J]. 林业科学, 2017,53(7):62-71.
[21]
敏张, 蔡瑞国, 李慧芝, 等. 盐胁迫环境下不同抗盐性小麦品种幼苗长势和内源激素的变化[J]. 生态学报, 2008,28(01):312-322.
[22]
徐磊, 王伟伟, 苏世超, 等. 小麦糖转运蛋白基因TaSWEET6的克隆与表达分析[J]. 麦类作物学报, 2016(11):1411-1418.
[23]
刘丹, 王建贺, 王从磊, 等. 不同浓度盐胁迫对小麦萌发和幼苗生长的影响[J]. 中国农学通报, 2016,32(24):49-54.
[24]
赵佩, 腾丽杰, 王轲. 小麦TaVIP1家族基因克隆、分子特性及功能分析[J]. 作物学报, 2017,043(2):201-209.
[25]
耿晓丽, 臧新山, 王飞, 等. 小麦耐热相关转录因子基因TabZIP28的分离及功能分析[J]. 农业生物技术学报, 2016,24(2):157-167.
碱性亮氨酸拉链(basic leucine zipper, bZIP)是植物中广泛存在的一类转录因子,参与多种胁迫响应与生长发育过程。本研究从小麦(Triticum aestivum)中克隆到一个热胁迫诱导的bZIP家族转录因子基因TabZIP28 (GenBank登录号: KT753298.1),ORF长度为1 713 bp,编码570个氨基酸。生物信息学分析结果表明,TabZIP28与拟南芥bZIP家族转录因子中B亚组的3个基因AtbZIP17、AtbZIP28 和AtbZIP49归为一类。氨基酸序列比对结果表明,该蛋白具有bZIP和跨膜结构域(transmembrane domain, TMD)两个保守结构域以及规范的位点1蛋白酶(site 1 protease, S1P)剪切位点。对该基因起始密码子ATG上游1 699 bp的序列进行顺式作用元件分析,发现该基因的启动子区域包含众多激素和逆境胁迫响应元件。通过qRT-PCR对该基因在逆境胁迫下的表达模式进行分析,结果表明,TabZIP28在热胁迫处理1 h即上调表达且达到最大值;用20% PEG 6000模拟干旱环境处理小麦幼苗后,TabZIP28在处理6 h达到最大值,并在12 h时急剧下降;对5 mmol/L H2O2处理响应比较缓慢,在处理12 h才上调表达;该基因不受到二硫苏糖醇(dithiothreitol, DTT)处理的诱导表达。在拟南芥(Arabidopsis thaliana)中过量表达TabZIP28基因,转基因株系在高温胁迫后的成活率和种子发芽率较野生型明显提高,说明该基因可能对植物的耐热性有贡献,可以作为耐热性育种的候选基因。
[26]
杨洪兵, 邱念伟, 陈敏, 等. 小麦耐盐机理及培育抗盐品种研究进展[J]. 山东师范大学学报:自然科学版, 2001,16(01):80-83.

RIGHTS & PERMISSIONS

Copyright reserved © 2020. Chinese Agricultural Association Bulletin. 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(1458 KB)

Accesses

Citation

Detail

Sections
Recommended

/