高温高湿下大豆钙依赖蛋白激酶基因在种子活力中的功能分析

发布时间：2018-05-03 01:01

本文选题：春大豆 + 钙依赖蛋白激酶(CDPK)　；参考：《南京农业大学》2016年博士论文

【摘要】：钙依赖蛋白激酶(calcium dependent protein kinases, CDPKs)普遍存在于植物和部分原生动物中,是植物特有的一类丝氨酸/苏氨酸型蛋白激酶,参与了多种Ca2+介导的信号通路,在植物发育信号和逆境信号转导中具有重要作用。大豆[Glycine max (L.) Merr.]是世界上重要的饲粮兼用作物,是植物蛋白的主要来源。南方春大豆区是我国大豆的主产区之一,该地区大豆种子发育成熟时期(R6期-R7期)以及收获期常会遇到高温高湿天气,发生种子田间劣变,导致种子活力下降。本课题组前期的差异蛋白质组学研究结果表明高温高湿胁迫后田间劣变抗性不同的春大豆种质中CDPK蛋白呈差异表达,qRT-PCR分析发现GmCDPKSK5在春大豆种子中存特异性高表达和田间劣变抗性差异表达。本研究在此基础上开展以下研究:(l)GmCDPKSK5互作蛋白的筛选与鉴定;(2)高温高湿胁迫下GmCDPKSK5及其互作蛋白基因在不同种子田间劣变抗性大豆种质种子中的表达分析;(3)GmCDPK Seed a基因的分离和GmCDPK Seed a蛋白的亚细胞定位;(4)高温高湿胁迫下,GmCDPK Seed a基因在不同种子活力大豆种质的表达模式分析;(5)GmCDPKSK5和GmCDPK Seed a过表达对拟南芥种子活力的影响。以期为进一步深入研究GmCDPK基因在参与大豆种子活力形成和响应逆境胁迫等方面的生物学功能奠定分子基础。主要研究结果如下:1.以GmCDPKSK5的ORF为诱饵对高温高湿胁迫下抗种子田间劣变春大豆种子膜蛋白酵母双杂交文库进行筛选和回转验证,初步获得了 6个与GmCDPKSK5互作的蛋白,分别是1个胚胎晚期丰富蛋白、1个翻译控制肿瘤蛋白、1个种子成熟蛋白、1个微粒体油酸脱氢酶以及2个未知功能蛋白;2.BiFC实验表明GmCDPKSK5与GmTCTP蛋白的互作主要发生在烟草叶片细胞膜;体外GST pull down实验进一步说明GST-GmCDPKSK5和His-GmTCTP融合蛋白在体外存在特异互作;荧光定量PCR实验发现:高温高湿胁迫下,在不抗种子田间劣变春大豆种质宁镇1号种子中,GwCDPKSK5基因与GmTCTP基因表达量均低于对照组,且在48 h、96h和168h处差异达极显著水平,在抗种子田间劣变春大豆种质湘豆3号中,GmCDPKSK5基因表达量在24h处显著高于对照组,在48h、96h和168 h处显著低于对照组,GmTCTP基因表达量在24 h处显著高于对照组,在48 h和168 h处显著低于对照组,而无论在对照还是高温高湿胁迫下,宁镇1号和湘豆3号的GmTCTP基因的表达量均显著高于GmCDPKSK5基因,且表达趋势一致,因此,推测GmCDPKSK5和GmTCTP共同参与高温高湿胁迫的调控。3.GmCDPK Seed a基因cDNA序列包含一个长1524 bp的开放阅读框(ORF),DNA序列长5333 bp,含有7个外显子和6个内含子;在GmCDPK Seed a基因5'上游2769 bp内分离到了该基因的启动子序列,其序列中存在热激响应(CCAATBOX1)、黄化诱导(ACGTATERD1)、低温诱导(LTRE1HVBLT49)和光调控(BOXCPSAS1、GATABOX)等顺式作用元件;GmCDPK Seed a基因编码的蛋白定位于细胞膜上。4.荧光定量PCR实验发现,高温高湿胁迫后,与对照相比在宁镇1号种子中GmCDPK Seed a基因下调表达,在湘豆3号种子中GmCDPK Seed a基因在24 h上调表达,其余时间点均下调表达,不同种子活力大豆种质在高温高湿胁迫后GmCDPK Seed a基因的表达量存在显著差异,在胁迫开始时,该基因在宁镇1号种子中的表达量略高于其在湘豆3号种子中的表达量,但随着胁迫时间的延长,其在湘豆3号中的表达量均显著高于宁镇1号,在168 h处,GmCDPK Seed a 基因在宁镇1号种子中的表达量略高于其在湘豆3号种子中的表达量。器官特异性表达实验结果表明:在湘豆3号中GmCDPK Seed a基因表达量从低至高依次为:盛荚期(R4)2cm长的幼英、茎、盛花期(R2)的花、根、叶、成熟种子,在大豆种子发育过程中GmCDPK Seed a基因表达量基本呈先上升后下降的过程,在开花后第20天表达量达到最大值。5.高温高湿胁迫下,GmCDPKSK5和GmCDPK Seed a过表达拟南芥种子发芽率高于cpk4-1突变体、WT、pBI121-GFP过表达拟南芥种子;cpk4-1突变体、WT、pBI121-GFP过表达、GmCDPKSK5过表达和GmCDPK Seed a过表达植株种子发芽势、发芽指数和活力指数均明显低于对照组,其中突变体植株种子发芽势、发芽指数和活力指数均为最低;而cpk4-1突变体、WT、pBI121-GFP过表达、GmCDPKSK5过表达和GmCDPK Seed a过表达植株种子的平均发芽天数较对照组均有所提高,以cpk4-1突变体植株种子提高幅度最大,GmCDPKS/K5过表达植株种子的平均发芽天数较WT、pBI121-GFP过表达种子高,GmCDPK Seed a过表达植株种子的平均发芽天数较WT、pBI121-GFP过表达种子低,说明GmCDPKSK5和GmCDPK Seed a均可以提高拟南芥种子活力。
[Abstract]:Calcium dependent protein kinases (CDPKs) is common in plants and parts of protozoa. It is a kind of plant specific serine / threonine type protein kinase, which is involved in a variety of Ca2+ mediated signaling pathways and plays an important role in plant development signal and stress signal transduction. Soybean [Glycine max (L.) Me Rr.] is an important crop in the world, and it is the main source of plant protein. The southern spring soybean region is one of the main producing areas of soybean in China. The mature period of soybean seed in the region (R6 phase -R7 stage) and the harvest period often meet high temperature and high humidity weather, the seed field deterioration and the seed vigor decline. The results of the hetero proteomics study showed that CDPK protein was expressed differently in the Spring Soybean Germplasm with different field variation resistance after high temperature and high humidity stress. QRT-PCR analysis found that GmCDPKSK5 was highly expressed in the seed of spring soybean and the differential expression of field deterioration resistance in spring soybean seeds. Based on this study, the following study was carried out: (L) GmCDPKSK5 interaction protein Screening and identification; (2) expression analysis of GmCDPKSK5 and its interaction protein gene in different seed field resistant soybean germplasm under high temperature and high humidity stress; (3) isolation of GmCDPK Seed a gene and subcellular localization of GmCDPK Seed a protein; (4) under high and high temperature and high humidity, GmCDPK Seed a gene in different seed vigor Soybean Germplasm Expression pattern analysis; (5) the effect of overexpression of GmCDPKSK5 and GmCDPK Seed a on seed vigor of Arabidopsis thaliana in order to lay a molecular basis for further in-depth study of the biological functions of GmCDPK gene in soybean seed vigor formation and response to stress stress. The main results are as follows: 1. with GmCDPKSK5 ORF as the bait for high temperature high Under wet stress, the seed membrane protein yeast two hybrid library for seed field deteriorated spring soybean seed was screened and proved by rotation. 6 proteins interacting with GmCDPKSK5 were preliminarily obtained, including 1 Advanced embryos rich proteins, 1 translation control tumor proteins, 1 seed mature proteins, 1 microsomal oleic dehydrogenases and 2 unknown functional eggs. 2.BiFC experiment showed that the interaction between GmCDPKSK5 and GmTCTP protein mainly occurred in the cell membrane of tobacco leaves; in vitro GST pull down experiment further indicated that the specific interaction between GST-GmCDPKSK5 and His-GmTCTP fusion protein existed in vitro; the fluorescence quantitative PCR experiment found that under high temperature and high humidity stress, it was in Ningzhen 1 In the seed, the expression of GwCDPKSK5 and GmTCTP genes were all lower than that in the control group, and the difference between 96h and 168h reached a very significant level at 48 h. The expression of GmCDPKSK5 gene was significantly higher in 24h than in the control group in the seed field deteriorated spring soybean germplasm Xiang bean 3, and was significantly lower in 48h, 96h and 168 h than in the control group, and the GmTCTP gene expression was 24 h. The location of the GmTCTP gene was significantly lower than the control group at 48 h and 168 h, and the expression of GmTCTP gene in Ningzhen 1 and xiangdou 3 was significantly higher than that of GmCDPKSK5 gene, and the expression trend was consistent with the control or high temperature and high humidity stress. Therefore, GmCDPKSK5 and GmTCTP were conjectured to participate in the regulation of.3.GmCDPK Seed under high temperature and humidity stress. The cDNA sequence of the a gene contains a 1524 BP open reading frame (ORF), the DNA sequence is 5333 BP long and contains 7 exons and 6 introns, and the promoter sequence of the gene is separated in the upstream 2769 BP of GmCDPK Seed a gene 5', and there are thermal shock response (CCAATBOX1), yellow induction, low temperature induction and light in the sequence of GmCDPK Seed a gene 5'. Regulation (BOXCPSAS1, GATABOX) and other cis acting elements; GmCDPK Seed a gene encoded protein located on the cell membrane.4. fluorescence quantitative PCR experiment found that after high temperature and high humidity stress, the GmCDPK Seed a gene in Ningzhen 1 was down regulated compared with the control, and the GmCDPK Seed gene was up to be expressed in 24 of xiangbean 3 seed and the rest time. The expression of GmCDPK Seed a gene in different seed vigor soybean germplasm has significant difference after high temperature and high humidity stress. The expression of this gene in Ningzhen 1 seed is slightly higher than that in the seed of xiangdou No. 3 at the beginning of stress, but the expression of the gene in xiangdou No. 3 is all with the prolongation of stress time. The expression of GmCDPK Seed a gene in Ningzhen No. 1 was slightly higher than that of Ningzhen No. 1 at 168 H. The experimental results of organ specific expression showed that the GmCDPK Seed a gene expression in xiangdou No. 3 from low to high was the young British, stem, flower, flower and root of the flourishing period (R4). During the development of soybean seeds, the expression of GmCDPK Seed a gene expression was first increased and then decreased during the development of soybean seeds. The twentieth days after flowering reached the maximum value of.5. high temperature and high humidity stress, GmCDPKSK5 and GmCDPK Seed a overexpressed the seed germination rate of Arabidopsis thaliana than cpk4-1 mutant, WT, pBI121-GFP overexpressed Arabidopsis seeds. Cpk4-1 mutant, WT, pBI121-GFP overexpression, GmCDPKSK5 overexpression and GmCDPK Seed a overexpressed seed germination potential, germination index and vigor index were significantly lower than the control group, among which the seed germination potential, germination index and vigor index were the lowest; cpk4-1 mutants, WT, pBI121-GFP overexpressed, GmCDPKSK5 overexpression and GmC. The average germination days of DPK Seed a overexpressed plant seeds were higher than those of the control group, and the maximum number of seeds increased with the cpk4-1 mutant. The average germination days of GmCDPKS/K5 overexpressed plant seeds were higher than that of WT, pBI121-GFP overexpressed seeds, and the average number of germination days of GmCDPK Seed a overexpressed plant seed was higher than that of WT, pBI121-GFP over expressed species. The results showed that GmCDPKSK5 and GmCDPK Seed a could improve the seed vigor of Arabidopsis thaliana.

【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S565.1

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