无核葡萄胚珠发育进程中EST的分析及败育相关基因的克隆
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摘要
无核葡萄食用方便,品质优良,是鲜食葡萄和制干葡萄的主要类型。生产中栽培的无核品种属于种子败育型,受精胚珠在发育途中败育而不能形成正常的种子,在果实成熟时仅留下大小不同的种子痕迹。无核葡萄胚珠的败育在不同年份、不同地点具有高度的稳定性,不受外界环境条件的影响,主要由无核葡萄的自身遗传基因所决定,因而具有十分重要的研究利用价值。
本研究以典型的无核品种‘无核白’败育前后胚珠为材料,构建cDNA文库,采用EST技术,并以文库为基础采用PCR技术克隆败育相关基因及其上游调控序列,以期获得无核葡萄败育相关的EST序列或基因;同时,分析有核葡萄‘京秀’和无核葡萄‘杨格尔’胚珠发育(败育)过程中活性氧的变化及其活性氧清除酶活性的变化,以揭示无核葡萄胚珠败育过程中活性氧代谢的变化趋势,探索无核葡萄胚珠发育过程中活性氧变化与种子败育的关系。研究主要取得如下结果:
1、提取不同发育时期胚珠总RNA,构建cDNA文库通过控制授粉,以花后27d、30d、33d、36d、39d、42d、45d、48d的‘无核白’胚珠为材料,采用改良SDS法提取总RNA,构建了胚珠败育前后不同时间点混合的cDNA文库。原始文库滴度为5.87×105 pfu/ml,有效库容量为5.55×105 pfu,重组率达96.60%。插入片段主要分布在0.5-2.0kb之间,平均大小为900bp左右。扩增文库滴度为2.135×109 pfu/ml。
2、文库的测序和EST分析通过随机测序,获得了214条质量好的EST序列。利用BlastN和BlastX程序将获得的214条序列与GenBank的nr数据库进行同源性分析,并将两种比较结果进行汇总,有144条序列与己知基因相似性很高,占67.29%;17条序列与功能尚未确定的假定蛋白或未知蛋白同源性很高,占7.94%;53条在nr数据库中未找到同源的序列,占24.77%。53条没有找到任何同源性的序列中,9条为葡萄新的EST序列。获得60个基因全长序列,文库中全长序列基因占有同源性基因161条的37.27%。其中167条EST在GenBank数据库的登陆号分别为EY254594、EY254595、EY254596、EY254922、EY254923、EY254924、EY254925、EY254926、EY254927、EY254928、EY254929、EY254930、EY254931、EY254932、EY254933、EY254934、EY254935、EY254936、EY254937、EY254938、EY254939、EY254940、EY254941、EY254942、EY254943、EY254944、EY254945、EY254946、EY254947、EY254948、EY254949、EY254950、EY254951、EY254952、EY254953、EY254954、EY254955、EY254956、EY254957、EY254958、EY254959、EY254960、EY254961、EY254962、EY254963、EY254964、EY254965、EY254966、EY254967、EY254968、EY254969、EY254970、EY254971、EY254972、EY254973、EY254974、EY254975、EY254976、EY254977、EY254978、EY254979、EY254980、EY254981、EY254982、EY254983、EY254984、EY254985、EY254986、EY254987、EY254988、EY254989、EY254990、EY254991、EY254992、EY254993、EY254994、EY254995、EY254996、EY254997、EY254998、EY254999、EY255000、EY255001、EY255002、EY255003、EY255004、EY255005、EY255006、EY255007、EY255008、EY255009、EY255010、EY255011、EY255012、EY255013、EY255014、EY255015、EY255016、EY255017、EY255018、EY255019、EY255020、EY255021、EY255022、EY255023、EY255024、EY255025、EY255026、EY255027、EY255028、EY255029、EY255030、EY255031、EY255032、EY255033、EY255034、EY255035、EY255036、EY255037、EY255038、EY255039、EY255040、EY255041、EY255042、EY255043、EY255044、EY255045、EY255046、EY255047、EY255048、EY255049、EY255050、EY255051、EY255052、EY255053、EY255054、EY255055、EY255056、EY255057、EY255058、EY255059、EY255060、EY255061、EY255062、EY255063、EY255064、EY255065、EY255066、EY255067、EY255068、EY255069、EY255070、EY255071、EY255072、EY255073、EY255074、EY255075、EY255076、EY255077、EY255078、EY255079、EY255080、EY255081、EY255082、EY255083、EY255084、EY255085。
3、活性氧代谢相关基因的克隆以构建的cDNA文库库液为模板,以载体两端通用引物和设计的特异引物通过PCR法扩增、拼接,获得葡萄锰超氧化物歧化酶(VvMnSOD)和葡萄抗坏血酸过氧化物酶(VvAPX)基因。通过文库直接测序获得葡萄金属硫蛋白基因(VvMT)、葡萄硫氧还蛋白基因(VvTRX)、葡萄脱氢抗坏血酸还原酶(VvDHAR)共5个活性氧代谢相关基因全长cDNA序列,进一步利用生物信息学软件对其进行序列特征分析。葡萄金属硫蛋白基因(VvMT)的cDNA序列长540bp,含有一个240bp的完整的开放阅读框架(ORF),编码的蛋白含有14个高比例的半胱氨酸(Cys)残基,占总氨基酸数的17. 72%,具有C-x-C、C-x-y-C和C-C的序列结构,在GenBank的登陆号为EU280163。同时,高度保守的半胱氨酸富含区(cysteine rich region, CR区)分布在蛋白质的两端,具有典型MT基因结构的三段式特征。克隆的葡萄金属硫蛋白可以归Type 2,是MT家族的第15个成员。该基因的DNA序列长998bp,包含3个外显子和2个内含子,在GenBank的登陆号为EU280165;通过酶切、接头连接、巢式PCR,克隆了783bp的葡萄金属硫蛋白基因的上游调控序列。其核心启动子区域位于658～708bp之间,序列中TATA-box、A-box、CAAT-box、G-Box、ARE、Box-4、CCGTCC-box、GAG-motif、HSE、Skn-1_motif、TC-rich repeats、repeatscircadian等12个可能的启动子上游调控元件,其中Skn-1_motif是胚乳中特异表达的保守序列。葡萄硫氧还蛋白基因(VvTRX)的cDNA长561bp,开放阅读框架(ORF) 381bp,编码126个氨基酸,在氨基酸的13-117之间有Thioredoxin保守结构域,活性功能位点WCXP[C/S]保守性很强,VvTrx属于硫氧还蛋白基因h型的3亚类,在GenBank的登陆号为EU280164。以基因组DNA为模板,扩增VvTrx基因DNA序列长989bp,包含3个外显子和2个内含子,在GenBank的登陆号为EU280166。VvMnSOD基因cDNA全长896bp,完整的开放阅读框架(ORF) 687bp,编码228个氨基酸,序列的189～196之间含有Mn和Fe超氧化物酶的金属结合结构域DvWEHAYY,聚类分析表明为MnSOD,在GenBank的登陆号为EU280161;VvAPX基因cDNA全长1044bp,完整的开放阅读框(ORF)长度为762bp,编码253个氨基酸,序列的19～226具有peroxidase结构域,4～249之间具有ascorbate_peroxidase保守结构域,33～44之间含一个过氧化物酶的活性位点(Peroxidases active site signature)序列为APLMLRLAWHSA;其编码的蛋白属于胞质抗坏血酸过氧化物酶,在GenBank的登陆号为EU280159;脱氢抗坏血酸还原酶(VvDHAR)cDNA长990bp,完整的开放阅读框架(ORF)长639bp,编码212个氨基酸,预测位于细胞质,为1类DHAR,在GenBank的登陆号为EU280162。
4、无核葡萄胚珠败育中蛋白质降解有关基因半胱氨酸蛋白酶及其抑制剂基因的克隆测序的VVTOV384的克隆3’端有poly(A)结构,5’端不完整。以文库菌液为模板,载体5端序列设计引物,采用半巢式PCR法进行5’RACE,克隆获得葡萄VvCysP基因,为木瓜蛋白酶型半胱氨酸蛋白酶,在GenBank的登陆号为EU280160,cDNA长1409bp,完整的开放阅读框架(ORF)长1134bp,编码377个氨基酸,是一个蛋白前体,成熟蛋白的长度可能为233个氨基酸残基,定位于细胞外。在氨基酸序列的163～174位点QGsCGSCWSfST为真核生物半胱氨酸蛋白酶的半胱氨酸活性位点,310～320位点LDHGVLLVGYG为真核生物半胱氨酸蛋白酶的组氨酸活性位点,334～353位点YWIiKNSWGENWGENGFYkI为真核生物半胱氨酸蛋白酶的天冬酰胺活性位点。半胱氨酸蛋白酶抑制剂基因(VvCPI)的cDNA长872bp,开放阅读框759bp,编码252个氨基酸,5～125为半胱氨酸蛋白酶抑制剂基因保守结构域CY,氨基酸序列的81～94位点EQVVAGKIYyLTLE为半胱氨酸蛋白酶抑制剂基因(CYSTATIN)活性位点,1～22位为信号肽;为一个蛋白前体,成熟蛋白质属于细胞外蛋白。
5、葡萄胚珠发育中活性氧和清除酶活性的变化以盛花后不同时期的无核葡萄品种杨格尔和有核葡萄品种京秀的胚珠为材料,测定胚珠中超氧阴离子( )和过氧化氢(H2O2)含量及活性氧清除酶活性的变化。结果表明:在两品种胚珠发育过程中均呈一直上升的趋势,在胚珠发育的中后期无核品种扬格尔产生速率明显大于有核品种京秀;胚珠中H2O2含量表现先升后降再升的趋势,但再升时无核品种扬格尔显著大于有核品种京秀;胚珠内活性氧清除酶超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(ASA-POD)及无核品种扬格尔的过氧化物酶(POD)表现先升后降的趋势,无核品种扬格尔的酶活性在胚珠败育后明显下降,有核品种京秀仍有较高的水平,且有核品种的POD活性先快速上升后一直保持缓慢上升的趋势。胚珠内活性氧类物质及活性氧清除酶类的变化趋势与无核葡萄杨格尔幼胚败育有一定的相关性。
Seedless grapevines are conveniently edible and with good quality. They are widely used for the fresh market and raisnins. Seedless grapevines in cultivation are seed abortive. The fertilized ovules cannot develop the normal seed and only form seed trace when fruit mature. The abortion of ovules are highly invariable and not affected by external factors at various times and places. It is under the control of genes in seedless grapevines, which are valuable for research and utilization.
In this study, a cDNA library was prepared from pre- and post abortive ovules of Thompson Seedless grapevines. EST and PCR techniques were used to clone the abortion-related genes and their up-stream regulatory sequences. Meanwhile, the activity of reactive oxygen species and reactive-oxygen-scavenging enzymes were analyzed in‘Jingxiu’and seedless grapevine‘Youngle’during ovules abortion/development, to reveal the trends of these enzymes and explore the relationship between the change of reactive oxygen species and seed abortion. The potential results are as follows:
1. RNA extraction and constructing cDNA library. At 27d, 30d, 33 d, 36d, 39d, 42d, 45d and 48d post-anthesis, ovules were collected from Thompson Seedless and snap frozen in liquid nitrogen. Total RNA was isolated respectively from above samples with SDS/phenol method modified partially. The pool of total RNA at various times was used to construct the cDNA library. The titer of primary library was 5.87×105 pfu/ml. The effective library capacity is 5.55×105 pfu. The recombinant percent accounted for 96.60%. The size of insert fragments basically ranged from 0.5 kb to 2.0 kb with average of 0.9 kb. The titer of the amplified library was 2.315×109 pfu/ml.
2. Sequencing and analysis of EST. 214 sequences with high quality were obtained. These sequences were searched in GenBank nr database for homologue using Blastn and Blastx programs and were further classified based on their functions. The results are as follows: 144 EST sequences accounting for 67.29% showed high homologue; 17 EST sequences accounting for 7.94% matched to the genes whose protein function were unclassified; 53 EST sequences accounting for 24.77% hit no any sequence in GenBank. 9 of the 53 represented novel ESTs. 60 full length sequences accounting for 28.03% of the library sequences were obtained. The 167 cDNA sequence have submitted to GenBank database, the GenBank accession numbers are as follows,EY254594、EY254595、EY254596、EY254922、EY254923、EY254924、EY254925、EY254926、EY254927、EY254928、EY254929、EY254930、EY254931、EY254932、EY254933、EY254934、EY254935、EY254936、EY254937、EY254938、EY254939、EY254940、EY254941、EY254942、EY254943、EY254944、EY254945、EY254946、EY254947、EY254948、EY254949、EY254950、EY254951、EY254952、EY254953、EY254954、EY254955、EY254956、EY254957、EY254958、EY254959、EY254960、EY254961、EY254962、EY254963、EY254964、EY254965、EY254966、EY254967、EY254968、EY254969、EY254970、EY254971、EY254972、EY254973、EY254974、EY254975、EY254976、EY254977、EY254978、EY254979、EY254980、EY254981、EY254982、EY254983、EY254984、EY254985、EY254986、EY254987、EY254988、EY254989、EY254990、EY254991、EY254992、EY254993、EY254994、EY254995、EY254996、EY254997、EY254998、EY254999、EY255000、EY255001、EY255002、EY255003、EY255004、EY255005、EY255006、EY255007、EY255008、EY255009、EY255010、EY255011、EY255012、EY255013、EY255014、EY255015、EY255016、EY255017、EY255018、EY255019、EY255020、EY255021、EY255022、EY255023、EY255024、EY255025、EY255026、EY255027、EY255028、EY255029、EY255030、EY255031、EY255032、EY255033、EY255034、EY255035、EY255036、EY255037、EY255038、EY255039、EY255040、EY255041、EY255042、EY255043、EY255044、EY255045、EY255046、EY255047、EY255048、EY255049、EY255050、EY255051、EY255052、EY255053、EY255054、EY255055、EY255056、EY255057、EY255058、EY255059、EY255060、EY255061、EY255062、EY255063、EY255064、EY255065、EY255066、EY255067、EY255068、EY255069、EY255070、EY255071、EY255072、EY255073、EY255074、EY255075、EY255076、EY255077、EY255078、EY255079、EY255080、EY255081、EY255082、EY255083、EY255084、EY255085.
3. Cloning the genes related to metabolism of reactive oxygen species. VvMnSOD and VvAPX genes were cloned using PCR with universal and specific primers and liquor of the library as template. The full length of five genes related to metabolism of reactive oxygen species, including VvMT, VvTRX, VvDHAR were obtained from library random sequencing. These sequences were analyzed using bioinformatics methods. The cDNA of VvMT was 540bp with an ORF of 240bp, which coded a protein with 14 Cys, accounting for 17.72% of total amino acid residues.The cDNA sequence have submitted to GenBank database, the GenBank accession numbers is EU280163. The protein sequence of VvMT contained C-x-C, C-x-y-C and C-C motifs and highly conserved cysteine rich region, which are the main features of MT genes. VvMT belonged to Type2 and the 15th member of MT family. The DNA sequence of VvMT was 998bp, including 3 exons and 2 introns. Its GenBank accession numbers is EU280165. Still, the 783bp up-stream regulatory sequence of VvMT was cloned using enzyme digest, adaptor ligation and nested PCR. The core promoter region spanned from 685bp to 708bp and contained 12 regulatory elements of TATA-box、A-box、CAAT-box、G-Box、ARE、Box-4、CCGTCC-box、GAG-motif、HSE、Skn-1_motif、TC-rich repeats、repeatscircadian. Skn-1_motif was the conserved sequence specifically expressed in endosperms.
The cDNA of VvTRX was 561bp with an ORF of 381bp coding a protein of 126 amino acid residues. Its GenBank accession numbers is EU280164. Thioredoxin spanned from 13 to 117 of the protein and the active site WCXP[C/S] was highly conserved. VvTrx belonged to the 3rd subfamily of thioredoxin h. The DNA sequence of VvTrx was 989bp, containing 3 exons and 2 introns. Its GenBank accession numbers is EU280166.
The cDNA of VvMnSOD, the member of MnSOD revealed by Cluster analysis was 896bp with an ORF of 687bp coding a protein of 228 amino acid residues. DvWEHAYY, the Mn and Fe binding domain of SOD spanned from 189 to 196 amino acid residues. Its GenBank accession numbers is EU280161.
The cDNA of VvAPX was 1044bp with an ORF of 762bp coding a protein of 253 amino acid residues. The peroxidase domain spanned from 19 to 226bp, ascorbate_peroxidase from 4 to 249bp and a Peroxidases active site signature, APLMLRLAWHSA from 33 to 44. VvAPX was one cytoplasmic ascorbate peroxidase. Its GenBank accession numbers is EU280159.
The cDNA of VvDHAR was 990bp with an ORF of 639bp coding a protein of 212 amino acid residues. VvDHAR belonged to 1st family of DHAR and probably located in cytoplasm. Its GenBank accession numbers is EU280162.
4. Cloning the CysP and CPI gene related to protein destination. The EST of VVTOV384 had full 3' poly (A) tail and not complete 5’end. VvCysP was cloned based on this EST using 5’RACE. The cDNA of VvCysP which was a cysteine proteinases of papain, was 1409bp with an ORF of 1134bp, coding a protein of 377 amino acid residues. This protein was a precursor and the mature one probably contained 233 amino acid residues and was an extracellular protein. QGsCGSCWSfST, spanned from 163 to 174 was the cysteine active site of cysteine protease in eukaryotes, LDHGVLLVGYG, spanned from 310 to 320 was the histidine active site, YWIiKNSWgenWGenGFYkI, spanned from 334 to 353 was the asparagine active site. Its GenBank accession numbers is EU280160.
The cDNA of VvCPI was 872bp with an ORF of 759bp coding a protein of 252 amino acid residues. CY domain of CYSTATIN spanned from 5 to 125, the active site, EQVVAGKIYyLTLE from 81 to 94 and the signal peptide from 1 to 22. This protein was a precursor and the mature one was an extracellular protein.
5. The change of the concentrations of reactive oxygen species and the activities of the antioxidant enzymes in the ovule of grape. The concentrations of and H2O2 and the activities of the antioxidant enzymes in the ovule of seedless grape‘Youngle’and seeded grape‘Jingxiu’were studied in the different periods after bloom. The results indicated that the concentrations of rose all the time, and the increasing rate in the ovule of seedless cultivar‘Youngle’was greater than that in the ovule of seeded cultivar‘Jingxiu’during middle and later periods of development; H2O2 concentration increased initially and decreased before it increased again. The subsequent increase in the seedless cultivar‘Youngle’was significantly greater than seeded cultivar‘Jingxiu’. The activities of SOD, CAT and AsA-POD increased initially and dropped drastically after ovule abortion in the seedless cultivar‘Youngle’. But it maintained high level in seeded cultivar‘Jingxiu’. The activity of POD in the ovule of the seedless cultivar‘Youngle’displayed a similar trend to the other enzymes, but the activity in seeded cultivar‘Jingxiu’increased constantly, but the increasing speed was lower in later stages. The changes of reactive oxygen content and the activity of antioxidant enzymes in ovules have some relevance to ovule abortion in the seedless cultivar‘Youngle’.
本研究以典型的无核品种‘无核白’败育前后胚珠为材料,构建cDNA文库,采用EST技术,并以文库为基础采用PCR技术克隆败育相关基因及其上游调控序列,以期获得无核葡萄败育相关的EST序列或基因;同时,分析有核葡萄‘京秀’和无核葡萄‘杨格尔’胚珠发育(败育)过程中活性氧的变化及其活性氧清除酶活性的变化,以揭示无核葡萄胚珠败育过程中活性氧代谢的变化趋势,探索无核葡萄胚珠发育过程中活性氧变化与种子败育的关系。研究主要取得如下结果:
1、提取不同发育时期胚珠总RNA,构建cDNA文库通过控制授粉,以花后27d、30d、33d、36d、39d、42d、45d、48d的‘无核白’胚珠为材料,采用改良SDS法提取总RNA,构建了胚珠败育前后不同时间点混合的cDNA文库。原始文库滴度为5.87×105 pfu/ml,有效库容量为5.55×105 pfu,重组率达96.60%。插入片段主要分布在0.5-2.0kb之间,平均大小为900bp左右。扩增文库滴度为2.135×109 pfu/ml。
2、文库的测序和EST分析通过随机测序,获得了214条质量好的EST序列。利用BlastN和BlastX程序将获得的214条序列与GenBank的nr数据库进行同源性分析,并将两种比较结果进行汇总,有144条序列与己知基因相似性很高,占67.29%;17条序列与功能尚未确定的假定蛋白或未知蛋白同源性很高,占7.94%;53条在nr数据库中未找到同源的序列,占24.77%。53条没有找到任何同源性的序列中,9条为葡萄新的EST序列。获得60个基因全长序列,文库中全长序列基因占有同源性基因161条的37.27%。其中167条EST在GenBank数据库的登陆号分别为EY254594、EY254595、EY254596、EY254922、EY254923、EY254924、EY254925、EY254926、EY254927、EY254928、EY254929、EY254930、EY254931、EY254932、EY254933、EY254934、EY254935、EY254936、EY254937、EY254938、EY254939、EY254940、EY254941、EY254942、EY254943、EY254944、EY254945、EY254946、EY254947、EY254948、EY254949、EY254950、EY254951、EY254952、EY254953、EY254954、EY254955、EY254956、EY254957、EY254958、EY254959、EY254960、EY254961、EY254962、EY254963、EY254964、EY254965、EY254966、EY254967、EY254968、EY254969、EY254970、EY254971、EY254972、EY254973、EY254974、EY254975、EY254976、EY254977、EY254978、EY254979、EY254980、EY254981、EY254982、EY254983、EY254984、EY254985、EY254986、EY254987、EY254988、EY254989、EY254990、EY254991、EY254992、EY254993、EY254994、EY254995、EY254996、EY254997、EY254998、EY254999、EY255000、EY255001、EY255002、EY255003、EY255004、EY255005、EY255006、EY255007、EY255008、EY255009、EY255010、EY255011、EY255012、EY255013、EY255014、EY255015、EY255016、EY255017、EY255018、EY255019、EY255020、EY255021、EY255022、EY255023、EY255024、EY255025、EY255026、EY255027、EY255028、EY255029、EY255030、EY255031、EY255032、EY255033、EY255034、EY255035、EY255036、EY255037、EY255038、EY255039、EY255040、EY255041、EY255042、EY255043、EY255044、EY255045、EY255046、EY255047、EY255048、EY255049、EY255050、EY255051、EY255052、EY255053、EY255054、EY255055、EY255056、EY255057、EY255058、EY255059、EY255060、EY255061、EY255062、EY255063、EY255064、EY255065、EY255066、EY255067、EY255068、EY255069、EY255070、EY255071、EY255072、EY255073、EY255074、EY255075、EY255076、EY255077、EY255078、EY255079、EY255080、EY255081、EY255082、EY255083、EY255084、EY255085。
3、活性氧代谢相关基因的克隆以构建的cDNA文库库液为模板,以载体两端通用引物和设计的特异引物通过PCR法扩增、拼接,获得葡萄锰超氧化物歧化酶(VvMnSOD)和葡萄抗坏血酸过氧化物酶(VvAPX)基因。通过文库直接测序获得葡萄金属硫蛋白基因(VvMT)、葡萄硫氧还蛋白基因(VvTRX)、葡萄脱氢抗坏血酸还原酶(VvDHAR)共5个活性氧代谢相关基因全长cDNA序列,进一步利用生物信息学软件对其进行序列特征分析。葡萄金属硫蛋白基因(VvMT)的cDNA序列长540bp,含有一个240bp的完整的开放阅读框架(ORF),编码的蛋白含有14个高比例的半胱氨酸(Cys)残基,占总氨基酸数的17. 72%,具有C-x-C、C-x-y-C和C-C的序列结构,在GenBank的登陆号为EU280163。同时,高度保守的半胱氨酸富含区(cysteine rich region, CR区)分布在蛋白质的两端,具有典型MT基因结构的三段式特征。克隆的葡萄金属硫蛋白可以归Type 2,是MT家族的第15个成员。该基因的DNA序列长998bp,包含3个外显子和2个内含子,在GenBank的登陆号为EU280165;通过酶切、接头连接、巢式PCR,克隆了783bp的葡萄金属硫蛋白基因的上游调控序列。其核心启动子区域位于658～708bp之间,序列中TATA-box、A-box、CAAT-box、G-Box、ARE、Box-4、CCGTCC-box、GAG-motif、HSE、Skn-1_motif、TC-rich repeats、repeatscircadian等12个可能的启动子上游调控元件,其中Skn-1_motif是胚乳中特异表达的保守序列。葡萄硫氧还蛋白基因(VvTRX)的cDNA长561bp,开放阅读框架(ORF) 381bp,编码126个氨基酸,在氨基酸的13-117之间有Thioredoxin保守结构域,活性功能位点WCXP[C/S]保守性很强,VvTrx属于硫氧还蛋白基因h型的3亚类,在GenBank的登陆号为EU280164。以基因组DNA为模板,扩增VvTrx基因DNA序列长989bp,包含3个外显子和2个内含子,在GenBank的登陆号为EU280166。VvMnSOD基因cDNA全长896bp,完整的开放阅读框架(ORF) 687bp,编码228个氨基酸,序列的189～196之间含有Mn和Fe超氧化物酶的金属结合结构域DvWEHAYY,聚类分析表明为MnSOD,在GenBank的登陆号为EU280161;VvAPX基因cDNA全长1044bp,完整的开放阅读框(ORF)长度为762bp,编码253个氨基酸,序列的19～226具有peroxidase结构域,4～249之间具有ascorbate_peroxidase保守结构域,33～44之间含一个过氧化物酶的活性位点(Peroxidases active site signature)序列为APLMLRLAWHSA;其编码的蛋白属于胞质抗坏血酸过氧化物酶,在GenBank的登陆号为EU280159;脱氢抗坏血酸还原酶(VvDHAR)cDNA长990bp,完整的开放阅读框架(ORF)长639bp,编码212个氨基酸,预测位于细胞质,为1类DHAR,在GenBank的登陆号为EU280162。
4、无核葡萄胚珠败育中蛋白质降解有关基因半胱氨酸蛋白酶及其抑制剂基因的克隆测序的VVTOV384的克隆3’端有poly(A)结构,5’端不完整。以文库菌液为模板,载体5端序列设计引物,采用半巢式PCR法进行5’RACE,克隆获得葡萄VvCysP基因,为木瓜蛋白酶型半胱氨酸蛋白酶,在GenBank的登陆号为EU280160,cDNA长1409bp,完整的开放阅读框架(ORF)长1134bp,编码377个氨基酸,是一个蛋白前体,成熟蛋白的长度可能为233个氨基酸残基,定位于细胞外。在氨基酸序列的163～174位点QGsCGSCWSfST为真核生物半胱氨酸蛋白酶的半胱氨酸活性位点,310～320位点LDHGVLLVGYG为真核生物半胱氨酸蛋白酶的组氨酸活性位点,334～353位点YWIiKNSWGENWGENGFYkI为真核生物半胱氨酸蛋白酶的天冬酰胺活性位点。半胱氨酸蛋白酶抑制剂基因(VvCPI)的cDNA长872bp,开放阅读框759bp,编码252个氨基酸,5～125为半胱氨酸蛋白酶抑制剂基因保守结构域CY,氨基酸序列的81～94位点EQVVAGKIYyLTLE为半胱氨酸蛋白酶抑制剂基因(CYSTATIN)活性位点,1～22位为信号肽;为一个蛋白前体,成熟蛋白质属于细胞外蛋白。
5、葡萄胚珠发育中活性氧和清除酶活性的变化以盛花后不同时期的无核葡萄品种杨格尔和有核葡萄品种京秀的胚珠为材料,测定胚珠中超氧阴离子( )和过氧化氢(H2O2)含量及活性氧清除酶活性的变化。结果表明:在两品种胚珠发育过程中均呈一直上升的趋势,在胚珠发育的中后期无核品种扬格尔产生速率明显大于有核品种京秀;胚珠中H2O2含量表现先升后降再升的趋势,但再升时无核品种扬格尔显著大于有核品种京秀;胚珠内活性氧清除酶超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(ASA-POD)及无核品种扬格尔的过氧化物酶(POD)表现先升后降的趋势,无核品种扬格尔的酶活性在胚珠败育后明显下降,有核品种京秀仍有较高的水平,且有核品种的POD活性先快速上升后一直保持缓慢上升的趋势。胚珠内活性氧类物质及活性氧清除酶类的变化趋势与无核葡萄杨格尔幼胚败育有一定的相关性。
Seedless grapevines are conveniently edible and with good quality. They are widely used for the fresh market and raisnins. Seedless grapevines in cultivation are seed abortive. The fertilized ovules cannot develop the normal seed and only form seed trace when fruit mature. The abortion of ovules are highly invariable and not affected by external factors at various times and places. It is under the control of genes in seedless grapevines, which are valuable for research and utilization.
In this study, a cDNA library was prepared from pre- and post abortive ovules of Thompson Seedless grapevines. EST and PCR techniques were used to clone the abortion-related genes and their up-stream regulatory sequences. Meanwhile, the activity of reactive oxygen species and reactive-oxygen-scavenging enzymes were analyzed in‘Jingxiu’and seedless grapevine‘Youngle’during ovules abortion/development, to reveal the trends of these enzymes and explore the relationship between the change of reactive oxygen species and seed abortion. The potential results are as follows:
1. RNA extraction and constructing cDNA library. At 27d, 30d, 33 d, 36d, 39d, 42d, 45d and 48d post-anthesis, ovules were collected from Thompson Seedless and snap frozen in liquid nitrogen. Total RNA was isolated respectively from above samples with SDS/phenol method modified partially. The pool of total RNA at various times was used to construct the cDNA library. The titer of primary library was 5.87×105 pfu/ml. The effective library capacity is 5.55×105 pfu. The recombinant percent accounted for 96.60%. The size of insert fragments basically ranged from 0.5 kb to 2.0 kb with average of 0.9 kb. The titer of the amplified library was 2.315×109 pfu/ml.
2. Sequencing and analysis of EST. 214 sequences with high quality were obtained. These sequences were searched in GenBank nr database for homologue using Blastn and Blastx programs and were further classified based on their functions. The results are as follows: 144 EST sequences accounting for 67.29% showed high homologue; 17 EST sequences accounting for 7.94% matched to the genes whose protein function were unclassified; 53 EST sequences accounting for 24.77% hit no any sequence in GenBank. 9 of the 53 represented novel ESTs. 60 full length sequences accounting for 28.03% of the library sequences were obtained. The 167 cDNA sequence have submitted to GenBank database, the GenBank accession numbers are as follows,EY254594、EY254595、EY254596、EY254922、EY254923、EY254924、EY254925、EY254926、EY254927、EY254928、EY254929、EY254930、EY254931、EY254932、EY254933、EY254934、EY254935、EY254936、EY254937、EY254938、EY254939、EY254940、EY254941、EY254942、EY254943、EY254944、EY254945、EY254946、EY254947、EY254948、EY254949、EY254950、EY254951、EY254952、EY254953、EY254954、EY254955、EY254956、EY254957、EY254958、EY254959、EY254960、EY254961、EY254962、EY254963、EY254964、EY254965、EY254966、EY254967、EY254968、EY254969、EY254970、EY254971、EY254972、EY254973、EY254974、EY254975、EY254976、EY254977、EY254978、EY254979、EY254980、EY254981、EY254982、EY254983、EY254984、EY254985、EY254986、EY254987、EY254988、EY254989、EY254990、EY254991、EY254992、EY254993、EY254994、EY254995、EY254996、EY254997、EY254998、EY254999、EY255000、EY255001、EY255002、EY255003、EY255004、EY255005、EY255006、EY255007、EY255008、EY255009、EY255010、EY255011、EY255012、EY255013、EY255014、EY255015、EY255016、EY255017、EY255018、EY255019、EY255020、EY255021、EY255022、EY255023、EY255024、EY255025、EY255026、EY255027、EY255028、EY255029、EY255030、EY255031、EY255032、EY255033、EY255034、EY255035、EY255036、EY255037、EY255038、EY255039、EY255040、EY255041、EY255042、EY255043、EY255044、EY255045、EY255046、EY255047、EY255048、EY255049、EY255050、EY255051、EY255052、EY255053、EY255054、EY255055、EY255056、EY255057、EY255058、EY255059、EY255060、EY255061、EY255062、EY255063、EY255064、EY255065、EY255066、EY255067、EY255068、EY255069、EY255070、EY255071、EY255072、EY255073、EY255074、EY255075、EY255076、EY255077、EY255078、EY255079、EY255080、EY255081、EY255082、EY255083、EY255084、EY255085.
3. Cloning the genes related to metabolism of reactive oxygen species. VvMnSOD and VvAPX genes were cloned using PCR with universal and specific primers and liquor of the library as template. The full length of five genes related to metabolism of reactive oxygen species, including VvMT, VvTRX, VvDHAR were obtained from library random sequencing. These sequences were analyzed using bioinformatics methods. The cDNA of VvMT was 540bp with an ORF of 240bp, which coded a protein with 14 Cys, accounting for 17.72% of total amino acid residues.The cDNA sequence have submitted to GenBank database, the GenBank accession numbers is EU280163. The protein sequence of VvMT contained C-x-C, C-x-y-C and C-C motifs and highly conserved cysteine rich region, which are the main features of MT genes. VvMT belonged to Type2 and the 15th member of MT family. The DNA sequence of VvMT was 998bp, including 3 exons and 2 introns. Its GenBank accession numbers is EU280165. Still, the 783bp up-stream regulatory sequence of VvMT was cloned using enzyme digest, adaptor ligation and nested PCR. The core promoter region spanned from 685bp to 708bp and contained 12 regulatory elements of TATA-box、A-box、CAAT-box、G-Box、ARE、Box-4、CCGTCC-box、GAG-motif、HSE、Skn-1_motif、TC-rich repeats、repeatscircadian. Skn-1_motif was the conserved sequence specifically expressed in endosperms.
The cDNA of VvTRX was 561bp with an ORF of 381bp coding a protein of 126 amino acid residues. Its GenBank accession numbers is EU280164. Thioredoxin spanned from 13 to 117 of the protein and the active site WCXP[C/S] was highly conserved. VvTrx belonged to the 3rd subfamily of thioredoxin h. The DNA sequence of VvTrx was 989bp, containing 3 exons and 2 introns. Its GenBank accession numbers is EU280166.
The cDNA of VvMnSOD, the member of MnSOD revealed by Cluster analysis was 896bp with an ORF of 687bp coding a protein of 228 amino acid residues. DvWEHAYY, the Mn and Fe binding domain of SOD spanned from 189 to 196 amino acid residues. Its GenBank accession numbers is EU280161.
The cDNA of VvAPX was 1044bp with an ORF of 762bp coding a protein of 253 amino acid residues. The peroxidase domain spanned from 19 to 226bp, ascorbate_peroxidase from 4 to 249bp and a Peroxidases active site signature, APLMLRLAWHSA from 33 to 44. VvAPX was one cytoplasmic ascorbate peroxidase. Its GenBank accession numbers is EU280159.
The cDNA of VvDHAR was 990bp with an ORF of 639bp coding a protein of 212 amino acid residues. VvDHAR belonged to 1st family of DHAR and probably located in cytoplasm. Its GenBank accession numbers is EU280162.
4. Cloning the CysP and CPI gene related to protein destination. The EST of VVTOV384 had full 3' poly (A) tail and not complete 5’end. VvCysP was cloned based on this EST using 5’RACE. The cDNA of VvCysP which was a cysteine proteinases of papain, was 1409bp with an ORF of 1134bp, coding a protein of 377 amino acid residues. This protein was a precursor and the mature one probably contained 233 amino acid residues and was an extracellular protein. QGsCGSCWSfST, spanned from 163 to 174 was the cysteine active site of cysteine protease in eukaryotes, LDHGVLLVGYG, spanned from 310 to 320 was the histidine active site, YWIiKNSWgenWGenGFYkI, spanned from 334 to 353 was the asparagine active site. Its GenBank accession numbers is EU280160.
The cDNA of VvCPI was 872bp with an ORF of 759bp coding a protein of 252 amino acid residues. CY domain of CYSTATIN spanned from 5 to 125, the active site, EQVVAGKIYyLTLE from 81 to 94 and the signal peptide from 1 to 22. This protein was a precursor and the mature one was an extracellular protein.
5. The change of the concentrations of reactive oxygen species and the activities of the antioxidant enzymes in the ovule of grape. The concentrations of and H2O2 and the activities of the antioxidant enzymes in the ovule of seedless grape‘Youngle’and seeded grape‘Jingxiu’were studied in the different periods after bloom. The results indicated that the concentrations of rose all the time, and the increasing rate in the ovule of seedless cultivar‘Youngle’was greater than that in the ovule of seeded cultivar‘Jingxiu’during middle and later periods of development; H2O2 concentration increased initially and decreased before it increased again. The subsequent increase in the seedless cultivar‘Youngle’was significantly greater than seeded cultivar‘Jingxiu’. The activities of SOD, CAT and AsA-POD increased initially and dropped drastically after ovule abortion in the seedless cultivar‘Youngle’. But it maintained high level in seeded cultivar‘Jingxiu’. The activity of POD in the ovule of the seedless cultivar‘Youngle’displayed a similar trend to the other enzymes, but the activity in seeded cultivar‘Jingxiu’increased constantly, but the increasing speed was lower in later stages. The changes of reactive oxygen content and the activity of antioxidant enzymes in ovules have some relevance to ovule abortion in the seedless cultivar‘Youngle’.
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