本文選題：春大豆 + 鈣依賴蛋白激酶(CDPK)�。� 參考：《南京農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：鈣依賴蛋白激酶(calcium dependent protein kinases, CDPKs)普遍存在于植物和部分原生動(dòng)物中,是植物特有的一類絲氨酸/蘇氨酸型蛋白激酶,參與了多種Ca2+介導(dǎo)的信號(hào)通路,在植物發(fā)育信號(hào)和逆境信號(hào)轉(zhuǎn)導(dǎo)中具有重要作用。大豆[Glycine max (L.) Merr.]是世界上重要的飼糧兼用作物,是植物蛋白的主要來源。南方春大豆區(qū)是我國大豆的主產(chǎn)區(qū)之一,該地區(qū)大豆種子發(fā)育成熟時(shí)期(R6期-R7期)以及收獲期常會(huì)遇到高溫高濕天氣,發(fā)生種子田間劣變,導(dǎo)致種子活力下降。本課題組前期的差異蛋白質(zhì)組學(xué)研究結(jié)果表明高溫高濕脅迫后田間劣變抗性不同的春大豆種質(zhì)中CDPK蛋白呈差異表達(dá),qRT-PCR分析發(fā)現(xiàn)GmCDPKSK5在春大豆種子中存特異性高表達(dá)和田間劣變抗性差異表達(dá)。本研究在此基礎(chǔ)上開展以下研究:(l)GmCDPKSK5互作蛋白的篩選與鑒定;(2)高溫高濕脅迫下GmCDPKSK5及其互作蛋白基因在不同種子田間劣變抗性大豆種質(zhì)種子中的表達(dá)分析;(3)GmCDPK Seed a基因的分離和GmCDPK Seed a蛋白的亞細(xì)胞定位;(4)高溫高濕脅迫下,GmCDPK Seed a基因在不同種子活力大豆種質(zhì)的表達(dá)模式分析;(5)GmCDPKSK5和GmCDPK Seed a過表達(dá)對(duì)擬南芥種子活力的影響。以期為進(jìn)一步深入研究GmCDPK基因在參與大豆種子活力形成和響應(yīng)逆境脅迫等方面的生物學(xué)功能奠定分子基礎(chǔ)。主要研究結(jié)果如下:1.以GmCDPKSK5的ORF為誘餌對(duì)高溫高濕脅迫下抗種子田間劣變春大豆種子膜蛋白酵母雙雜交文庫進(jìn)行篩選和回轉(zhuǎn)驗(yàn)證,初步獲得了 6個(gè)與GmCDPKSK5互作的蛋白,分別是1個(gè)胚胎晚期豐富蛋白、1個(gè)翻譯控制腫瘤蛋白、1個(gè)種子成熟蛋白、1個(gè)微粒體油酸脫氫酶以及2個(gè)未知功能蛋白;2.BiFC實(shí)驗(yàn)表明GmCDPKSK5與GmTCTP蛋白的互作主要發(fā)生在煙草葉片細(xì)胞膜;體外GST pull down實(shí)驗(yàn)進(jìn)一步說明GST-GmCDPKSK5和His-GmTCTP融合蛋白在體外存在特異互作;熒光定量PCR實(shí)驗(yàn)發(fā)現(xiàn):高溫高濕脅迫下,在不抗種子田間劣變春大豆種質(zhì)寧鎮(zhèn)1號(hào)種子中,GwCDPKSK5基因與GmTCTP基因表達(dá)量均低于對(duì)照組,且在48 h、96h和168h處差異達(dá)極顯著水平,在抗種子田間劣變春大豆種質(zhì)湘豆3號(hào)中,GmCDPKSK5基因表達(dá)量在24h處顯著高于對(duì)照組,在48h、96h和168 h處顯著低于對(duì)照組,GmTCTP基因表達(dá)量在24 h處顯著高于對(duì)照組,在48 h和168 h處顯著低于對(duì)照組,而無論在對(duì)照還是高溫高濕脅迫下,寧鎮(zhèn)1號(hào)和湘豆3號(hào)的GmTCTP基因的表達(dá)量均顯著高于GmCDPKSK5基因,且表達(dá)趨勢(shì)一致,因此,推測(cè)GmCDPKSK5和GmTCTP共同參與高溫高濕脅迫的調(diào)控。3.GmCDPK Seed a基因cDNA序列包含一個(gè)長1524 bp的開放閱讀框(ORF),DNA序列長5333 bp,含有7個(gè)外顯子和6個(gè)內(nèi)含子;在GmCDPK Seed a基因5'上游2769 bp內(nèi)分離到了該基因的啟動(dòng)子序列,其序列中存在熱激響應(yīng)(CCAATBOX1)、黃化誘導(dǎo)(ACGTATERD1)、低溫誘導(dǎo)(LTRE1HVBLT49)和光調(diào)控(BOXCPSAS1、GATABOX)等順式作用元件;GmCDPK Seed a基因編碼的蛋白定位于細(xì)胞膜上。4.熒光定量PCR實(shí)驗(yàn)發(fā)現(xiàn),高溫高濕脅迫后,與對(duì)照相比在寧鎮(zhèn)1號(hào)種子中GmCDPK Seed a基因下調(diào)表達(dá),在湘豆3號(hào)種子中GmCDPK Seed a基因在24 h上調(diào)表達(dá),其余時(shí)間點(diǎn)均下調(diào)表達(dá),不同種子活力大豆種質(zhì)在高溫高濕脅迫后GmCDPK Seed a基因的表達(dá)量存在顯著差異,在脅迫開始時(shí),該基因在寧鎮(zhèn)1號(hào)種子中的表達(dá)量略高于其在湘豆3號(hào)種子中的表達(dá)量,但隨著脅迫時(shí)間的延長,其在湘豆3號(hào)中的表達(dá)量均顯著高于寧鎮(zhèn)1號(hào),在168 h處,GmCDPK Seed a 基因在寧鎮(zhèn)1號(hào)種子中的表達(dá)量略高于其在湘豆3號(hào)種子中的表達(dá)量。器官特異性表達(dá)實(shí)驗(yàn)結(jié)果表明:在湘豆3號(hào)中GmCDPK Seed a基因表達(dá)量從低至高依次為:盛莢期(R4)2cm長的幼英、莖、盛花期(R2)的花、根、葉、成熟種子,在大豆種子發(fā)育過程中GmCDPK Seed a基因表達(dá)量基本呈先上升后下降的過程,在開花后第20天表達(dá)量達(dá)到最大值。5.高溫高濕脅迫下,GmCDPKSK5和GmCDPK Seed a過表達(dá)擬南芥種子發(fā)芽率高于cpk4-1突變體、WT、pBI121-GFP過表達(dá)擬南芥種子;cpk4-1突變體、WT、pBI121-GFP過表達(dá)、GmCDPKSK5過表達(dá)和GmCDPK Seed a過表達(dá)植株種子發(fā)芽勢(shì)、發(fā)芽指數(shù)和活力指數(shù)均明顯低于對(duì)照組,其中突變體植株種子發(fā)芽勢(shì)、發(fā)芽指數(shù)和活力指數(shù)均為最低;而cpk4-1突變體、WT、pBI121-GFP過表達(dá)、GmCDPKSK5過表達(dá)和GmCDPK Seed a過表達(dá)植株種子的平均發(fā)芽天數(shù)較對(duì)照組均有所提高,以cpk4-1突變體植株種子提高幅度最大,GmCDPKS/K5過表達(dá)植株種子的平均發(fā)芽天數(shù)較WT、pBI121-GFP過表達(dá)種子高,GmCDPK Seed a過表達(dá)植株種子的平均發(fā)芽天數(shù)較WT、pBI121-GFP過表達(dá)種子低,說明GmCDPKSK5和GmCDPK Seed a均可以提高擬南芥種子活力。
[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.

【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S565.1

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