水稻胚乳发育相关基因的筛选及功能分析
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摘要
水稻在我国粮食生产中具有举足轻重的作用。除其经济价值重要外,水稻转化体系成熟、基因组相对较小、全基因组序列测序已完成,水稻已成为单子叶植物分子遗传研究的重要模式植物。
稻米产量和品质的形成是个非常复杂的生理过程。胚乳是人们直接食用的部分,占精米的90%以上,胚乳发育直接影响着水稻的产量和品质。因此水稻胚乳发育相关基因的研究无论在基础理论还是在实际应用上都具有重要意义。
首先,本研究利用水稻T-DAN插入启动子捕获标签,构建了25,000个水稻T-DNA插入启动子捕获突变体群体,对其中2,653个水稻T-DNA插入启动子捕获系进行了GUS(β-glucuronidase)报道基因表达检测,获得了GUS在各组织器官表达的63个水稻T-DNA插入启动子捕获系,其中水稻胚乳发育相关的T-DNA插入启动子捕获系24个,并对一个胚乳特异表达启动子捕获系进行了初步研究,这将为利用T-DNA插入启动子捕获系进行胚乳发育相关基因研究提供材料基础和理论依据。
其次,本研究根据转录因子spf1序列同源克隆了大麦胚乳发育相关转录因子Susiba2(Sugar signaling in barley)以及水稻OsSusiba2基因,水稻OsSusiba2基因与大麦Susiba2基因有80%同源。本研究利用反义RNA和RNAi技术,构建OsSusiba2基因反义结构表达载体、OsSusiba2基因RNAi干扰表达载体及大麦Susiba2基因的过表达载体,用农杆菌介导法将它们导入水稻,利用其后代研究它们对水稻胚乳发育及淀粉代谢的影响。
其主要研究内容和结果如下:
一、水稻胚乳发育相关启动子捕获系的筛选
1.构建启动子捕获植物表达载体pCAMBIA1300GUSA-Hyg,利用农杆菌介导法将其导入籼稻明恢86,获得了25,000个水稻T-DNA插入启动子捕获系。对2,872个T0代水稻启动子捕获系进行了标记基因潮霉素磷酸转移酶表达筛选,其中
2,653个为阳性转基因水稻株系,阳性率达到92.4%。
2.对2,653个水稻启动子捕获系进行了两次GUS报告基因表达检测表明:共有63个水稻启动子捕获系在其不同时期、不同器官包括根、茎、叶、颖花、胚、胚乳等组织中检测到GUS表达,占总数2.37%;其中GUS活性为胚乳倾向或优势表达的水稻启动子捕获系24个,占总数0.90%;GUS活性在胚中表达的水稻启动子捕获系9个,占总数0.34%;GUS活性在花药中表达的水稻启动子捕获系为20个,占检测总数0.70%,而在根、茎、叶和颖壳中表达分别为6个、18个、7个和5个。
3.对63个部分T2代水稻启动子捕获系抗性基因分离比分析表明:39个中有28个水稻启动子捕获系为单T-DNA位点插入,占71.8%。Southern blot分析表明,水稻启动子捕获系w9101,w9106,w9154均为单拷贝。通过Tail-PCR扩增获得6个水稻胚乳发育相关启动子捕获系的侧翼序列并分析了插入位点详细信息。
二、一个水稻胚乳特异表达启动子捕获系的初步分析
1.对Tail-PCR扩增获得的水稻胚乳特异表达启动子捕获系w9101的侧翼序列分析发现,该T-DNA插入在水稻第6号染色体AP004750克隆的基因P0421H01.22和基因P0421H01.23之间,由于gus基因与基因P0421H01.23表达方向相同,表明w9101中GUS表达可能是P0421H01.23基因的启动子所起的。生物信息学分析表明: P0421H01.23基因编码一个由601个氨基酸组成的蛋白质,是一个肽转运子,编码的蛋白很可能是一个疏水性肽转运蛋白,跨膜区域和CHL1一样有12个α-螺旋跨膜区,这是肽转运蛋白家族的普遍特征,因此可推测P0421H01.23基因就是一个肽转运子(暂命名为OsPtr1);进一步用生物信息学软件分析其启动子发现,它是一个具有胚乳特异性表达活性特征的启动子。
2.RT-PCR结果分析表明:OsPtr1基因是一个胚乳特异性表达基因,并且T-DNA的插入使得OsPtr1基因功能缺失。OsPtr1基因和GFP融合表达亚细胞定位于质膜上。
3.分离了OsPtr1基因的cDNA全长,并构建OsPtr1基因的过表达载体Gt1-OsPtr1,用农杆菌介导法获得其转基因水稻植株,southern blot分析表明OsPtr1基因已经整合到水稻基因组。
4.稻米氮代谢相关物质含量分析表明:T-DNA插入使得启动子捕获系w9101的蛋白质含量及氨基酸含量都有一定程度的降低。和对照相比,三个转过表达OsPtr1基因水稻植株的蛋白质总量、17种氨基酸含量及氨基酸总量均有不同程度的增加。其中半必需氨基酸中精氨酸、6种必需氨基酸、谷氨酸、天门冬氨酸、蛋白质总量及氨基酸总量有较明显提高。
三、Susiba2和OsSusiba2基因在水稻淀粉代谢调控中的功能分析
1.构建OsSusiba2基因RNAi干扰和反义RNA植物表达载体,构建Susiba2基因过表达植物表达载体。获得以上三个植物表达载体的T4代转基因水稻纯合株系。
2.转基因水稻植株southern blot分析表明:Susiba2基因过表达转基因水稻株系(SRB77和SRB80)以及OsSusiba2基因RNAi干扰转基因水稻株系SRI90为两个拷贝,OsSusiba2基因反义RNA转基因水稻株系(SRR11和SRR39)以及OsSusiba2基因RNAi干扰转基因水稻株系SRI86为单拷贝。
3.转基因水稻植株northern blot分析表明:与对照相比,转反义OsSusiba2基因及其RNAi干扰的水稻株系内源淀粉分支酶表现出一定程度的抑制。转过表达Susiba2基因水稻株系内源淀粉分支酶表现出一定程度增强。
4.糖诱导下转基因水稻的表达分析表明:在蔗糖和甘露醇处理下,野生型、转反义OsSusiba2基因水稻株系和转RNAi干扰OsSusiba2基因水稻株系叶片的内源淀粉分支酶表达基本没有什么变化外,转过表达大麦Susiba2基因水稻株系的蔗糖处理要比甘露醇处理的内源淀粉分支酶表达有所提高,说明Susiba2基因和OsSusiba2基因均受蔗糖诱导表达,而不受甘露醇的影响。
5.转基因稻米直链淀粉含量分析表明:对照和转过表达Susiba2基因水稻的直链淀粉含量变化不大,转反义OsSusiba2基因及其RNAi干扰OsSusiba2基因水稻株系的直链淀粉含量均有明显下降的趋势,特别是OsSusiba2基因反义RNA转基因水稻株系SRR11和OsSusiba2基因RNAi干扰转基因水稻株系SRI86降幅较大。
6.转基因水稻植株表型分析表明:对照和转过表达Susiba2基因水稻在表型上基本接近;转RNAi干扰OsSusiba2基因水稻株系和前两种相比无效穗数增多,而引起结实率、实粒数等下降;转反义OsSusiba2基因水稻株系则下降更为明显。
7.转基因水稻的产量性状分析表明:转反义OsSusiba2基因及RNAi干扰OsSusiba2基因水稻株系比对照及过表达Susiba2基因水稻株系的单株瘪粒数明显增多,而单株实粒数和单株实粒重则明显减少,千粒重则无明显变化;转过表达Susiba2基因水稻株系和对照相比无较大变化;转反义OsSusiba2基因及RNAi干扰OsSusiba2基因水稻株系比过表达Susiba2基因水稻株系的单株穗数有明显的增多,而结实率和穗实粒数则明显减少。
8.转基因水稻碳水化合物分配分析表明:转过表达Susiba2基因水稻根茎叶干重比较对照、转RNAi干扰OsSusiba2基因水稻株系和转反义OsSusiba2基因水稻株系均有所提高,说明转过表达Susiba2基因水稻可表现为更高的生物学产量。
Rice is an important food crop in china.With the completion of rice genome sequencing,and it has been as an important monocotyledonous model species of molecular genetics studies for relate small genome and high efficient transformation system.
Formation of rice production and quality is a very complex physiological processes. Food for people is endosperm, which about 90% of milled rice, endosperm in rice directly influences on the yield and quality. so studying on genes of rice endosperm-related development will have great significance both basic theory and practical application.
Firstly, In this study, we will build rice promoter trap system by T-DAN tags, Obtaining 25,000 rice promoter trap lines, 63 of rice promoter trap lines were expressed by screening GUS expression from 2,653 lines, Including 24 endosperm-related lines, and preliminary function analysis of a rice promoter trap line of endosperm-related development, It will provide material and theoretical basis for studying on endosperm-related development gene by promoter trap system of T-DNA tagging.
Secondly, Transcription factor Susiba2 (Sugar signaling in barley) related to barley endosperm development and rice OsSusiba2 gene were cloned based on transcription factor spf1 sequences, and it has 80% homology between Susiba2 and OsSusiba2. In this study, it will construct antisense RNA vector of OsSusiba2 gene, RNAi vector of OsSusiba2 gene and overexpression vector of barley Susiba2 respectively.harboring them into rice gemone by Agrobacterium-mediated, Studies on endosperm development and starch metabolism in rice by their offspring.
Main contents and results are as follows:
Part I: Screening rice promoter trap lines of endosperm-related development
1.The binary vector pCAMBIA1300GUSA-Hyg contained the promoterless β-glucuronidase (GUS) was introdued into Oryza sativa L. Minghui 86 by Agrobacterium-mediated transformation, Obtaining 25,000 rice promoter trap lines. Hygromycin phosphotransferase expression analysis of 2,872 rice promoter trap lines of T0 generation showed that 2,653 of them were positive, the positive rate of 92.4%.
2. GUS histochemical assay of 2,653 rice promoter trap lines for two times was carried out in its differential organ at differential stage, including root, stem, leave, spekilet, embryo and emdospern. The data revealed that 63 rice promoter lines were GUS positive ,accounting for 2.73%; 24 rice promoter trap lines were expressed in endosperm, 0.90% of total number tested; nine rice promoter trap lines were expressed GUS activity in embryo, 0.34% of total number tested; 20 rice promoter trap lines were expressed in anther , accounting for 0.70% ; and five, eighteen, seven, five promoter trap lines were expressed in the roots, stem, leaves, hulls respectively.
3.Segregation ratio of hygromycin Phosphotransferase expression revealed that 28 rice promoter lines contained one loci T-DNA inserted from 39 lines, 71.8% among them.Southern blot analysis of the rice promoter trapping lines showed that w9101, w9106 and w9154 are single T-DNA inserted. Obtaining franking sequences of six endosperm-related lines by Tail-PCR and its deTail information of candidate genes .
PartⅡ: Preliminary analysis of rice endosperm-specific promoter trapping line
1.Obtained flanking sequence of endosperm-specific expression promoter trap line w9101 by TAIL-PCR, whom T-DNA was inserted between P0421H01.22 and P0421H01.23 of AP004750 in chromosome 6 by flanking sequence analysis, and P0421H01.23 is the same order with GUS, indicating that GUS activity was expressed by the promoter of P0421H01.23. Bioinformatics analysis shows that P0421H01.23 gene encods a 601 amino acid protein, it may be a hydrophobic peptide transporter, including 12α- helix transmembrane domain like CHL1, which is common characteristics of peptide transporter family, it can be speculated that P0421H01.23 is a peptide transporter gene( temporarily named OsPtr1) . Its promoter has a feature of endosperm-specific expression activity by bioinformatics softwares .
2. RT-PCR analysis showed that OsPtr1was endosperm-specific expression , OsPtr1 was knockouted by T-DNA inseted.OsPtr1-GFP fusion protein was espressed only in plastid membrane by subcellular localization.
3.Full-length cDNA of OsPtr1 was isolated , and overexpression vector Gt1-OsPtr1 was constructed, transgenic rice plantlets of the vector was obtained by Agrobacterium-mediated. OsPtr1 gene has integrated into rice genome by southern blot analysis.
4.Substance content analysis in rice of nitrogen metabolism-related showed that:Protein and amino acid content of promoter trap line w9101 were reduced .Total amino acid ,17 kinds of amino acids of transgenic rice plants of over-expression OsPtr1 have been increased at different levels compared to the control. Arginine among semi-indispensable amino acid ,6 kinds of indispensable amino acid,glutamic acid, aspartic acid, total proteins and amino acids of them are increased obviously.
Part III: Function analysis of Susiba2 and OsSusiba2 genes regulating starch metabolism in rice
1.Constructing vectors of RNAi and antisense RNA of OsSusiba2 gene, over-expression vector of Susiba2 gene. Obtaining T4 generation homozygous of transgenic rice for above three vectors.
2.Southern blot analysis of transgenic rice plantlets showed that two T-DNA were inserted into transgenic lines of over-expression Susiba2(SRB77and SRB)and transgenic line of OsSusiba2 gene for RNAi(SRI90) ; While transgenic lines of OsSusiba2 gene for antisense RNA (SRR11 and SRR39), transgenic line of OsSusiba2 gene for RNAi(SRI86) have only one T-DNA inserted.
3. Northern blot analysis of transgenic rice plantlets showed that: endogenous starch branching enzyme of transgenic rice for RNAi and antisense RNA of OsSusiba2 gene were suppressed and endogenous starch branching enzyme of transgenic rice for over-expression Susiba2 were inproved compared to wt .
4. Northern blot analysis of transgenic rice induced in sugar showed that wild type, transgenic rice of antisense and RNAi OsSusiba2 gene,of which endogenous starch branching enzyme of rice leaves dipped into sugar and manitol were unchanged.but endogenous starch branching enzyme of rice leaves of over-expression Susiba2 dipped in sugar were increased ,compare to in manitol. These indicated that OsSusiba2 and Susiba2 were induced in sugar, and not induced in manitol.
5. Amylose content of transgenic rice analysis showed that amylose content of transgenic rice of antisense and RNAi OsSusiba2 were decreased significantly compare to wt and transgenic rice of over-expression susiba2, Transgenic lines of OsSusiba2 gene for antisense RNA(SRR11) and transgenic line of OsSusiba2 gene for RNAi (SRI86) were decreased most apparently ,while amylose content of wt and transgenic rice of over-expression susiba2 is like .
6. Phenotype analysis of transgenic plantlets showed that spikes number per transgenic plantlets of antisense and RNAi OsSusiba2 were increased significantly compare to wt and transgenic rice of over-expression susiba2. while setting rate , grain filled number of them were decreased apparently. but these phenotypes of wt and transgenic rice of over-expression susiba2 were close on the phenotype.
7. Yield characterics of transgenic rice analysis showed that empty grains and panicles per transgenic plantlets of antisense and RNAi OsSusiba2 gene were increased significantly compare to wt and transgenic rice of over-expression susiba2, and grains filled number, weight of grain filled , seed setting rate, grain filled number per panicle of them is reduced significantly,1000 grain weight of them did not change significantly.
8. Distribution of carbohydrate of transgenic rice analysis shows that dry weight of roots, stems,leaves of transgenic rice of over-expression susiba2 were improved compare to wt, transgenic rice plantlets of antisense and RNAi OsSusiba2 gene , indicating that biological yield of transgenic rice of over-expression susiba2 were improved.
稻米产量和品质的形成是个非常复杂的生理过程。胚乳是人们直接食用的部分,占精米的90%以上,胚乳发育直接影响着水稻的产量和品质。因此水稻胚乳发育相关基因的研究无论在基础理论还是在实际应用上都具有重要意义。
首先,本研究利用水稻T-DAN插入启动子捕获标签,构建了25,000个水稻T-DNA插入启动子捕获突变体群体,对其中2,653个水稻T-DNA插入启动子捕获系进行了GUS(β-glucuronidase)报道基因表达检测,获得了GUS在各组织器官表达的63个水稻T-DNA插入启动子捕获系,其中水稻胚乳发育相关的T-DNA插入启动子捕获系24个,并对一个胚乳特异表达启动子捕获系进行了初步研究,这将为利用T-DNA插入启动子捕获系进行胚乳发育相关基因研究提供材料基础和理论依据。
其次,本研究根据转录因子spf1序列同源克隆了大麦胚乳发育相关转录因子Susiba2(Sugar signaling in barley)以及水稻OsSusiba2基因,水稻OsSusiba2基因与大麦Susiba2基因有80%同源。本研究利用反义RNA和RNAi技术,构建OsSusiba2基因反义结构表达载体、OsSusiba2基因RNAi干扰表达载体及大麦Susiba2基因的过表达载体,用农杆菌介导法将它们导入水稻,利用其后代研究它们对水稻胚乳发育及淀粉代谢的影响。
其主要研究内容和结果如下:
一、水稻胚乳发育相关启动子捕获系的筛选
1.构建启动子捕获植物表达载体pCAMBIA1300GUSA-Hyg,利用农杆菌介导法将其导入籼稻明恢86,获得了25,000个水稻T-DNA插入启动子捕获系。对2,872个T0代水稻启动子捕获系进行了标记基因潮霉素磷酸转移酶表达筛选,其中
2,653个为阳性转基因水稻株系,阳性率达到92.4%。
2.对2,653个水稻启动子捕获系进行了两次GUS报告基因表达检测表明:共有63个水稻启动子捕获系在其不同时期、不同器官包括根、茎、叶、颖花、胚、胚乳等组织中检测到GUS表达,占总数2.37%;其中GUS活性为胚乳倾向或优势表达的水稻启动子捕获系24个,占总数0.90%;GUS活性在胚中表达的水稻启动子捕获系9个,占总数0.34%;GUS活性在花药中表达的水稻启动子捕获系为20个,占检测总数0.70%,而在根、茎、叶和颖壳中表达分别为6个、18个、7个和5个。
3.对63个部分T2代水稻启动子捕获系抗性基因分离比分析表明:39个中有28个水稻启动子捕获系为单T-DNA位点插入,占71.8%。Southern blot分析表明,水稻启动子捕获系w9101,w9106,w9154均为单拷贝。通过Tail-PCR扩增获得6个水稻胚乳发育相关启动子捕获系的侧翼序列并分析了插入位点详细信息。
二、一个水稻胚乳特异表达启动子捕获系的初步分析
1.对Tail-PCR扩增获得的水稻胚乳特异表达启动子捕获系w9101的侧翼序列分析发现,该T-DNA插入在水稻第6号染色体AP004750克隆的基因P0421H01.22和基因P0421H01.23之间,由于gus基因与基因P0421H01.23表达方向相同,表明w9101中GUS表达可能是P0421H01.23基因的启动子所起的。生物信息学分析表明: P0421H01.23基因编码一个由601个氨基酸组成的蛋白质,是一个肽转运子,编码的蛋白很可能是一个疏水性肽转运蛋白,跨膜区域和CHL1一样有12个α-螺旋跨膜区,这是肽转运蛋白家族的普遍特征,因此可推测P0421H01.23基因就是一个肽转运子(暂命名为OsPtr1);进一步用生物信息学软件分析其启动子发现,它是一个具有胚乳特异性表达活性特征的启动子。
2.RT-PCR结果分析表明:OsPtr1基因是一个胚乳特异性表达基因,并且T-DNA的插入使得OsPtr1基因功能缺失。OsPtr1基因和GFP融合表达亚细胞定位于质膜上。
3.分离了OsPtr1基因的cDNA全长,并构建OsPtr1基因的过表达载体Gt1-OsPtr1,用农杆菌介导法获得其转基因水稻植株,southern blot分析表明OsPtr1基因已经整合到水稻基因组。
4.稻米氮代谢相关物质含量分析表明:T-DNA插入使得启动子捕获系w9101的蛋白质含量及氨基酸含量都有一定程度的降低。和对照相比,三个转过表达OsPtr1基因水稻植株的蛋白质总量、17种氨基酸含量及氨基酸总量均有不同程度的增加。其中半必需氨基酸中精氨酸、6种必需氨基酸、谷氨酸、天门冬氨酸、蛋白质总量及氨基酸总量有较明显提高。
三、Susiba2和OsSusiba2基因在水稻淀粉代谢调控中的功能分析
1.构建OsSusiba2基因RNAi干扰和反义RNA植物表达载体,构建Susiba2基因过表达植物表达载体。获得以上三个植物表达载体的T4代转基因水稻纯合株系。
2.转基因水稻植株southern blot分析表明:Susiba2基因过表达转基因水稻株系(SRB77和SRB80)以及OsSusiba2基因RNAi干扰转基因水稻株系SRI90为两个拷贝,OsSusiba2基因反义RNA转基因水稻株系(SRR11和SRR39)以及OsSusiba2基因RNAi干扰转基因水稻株系SRI86为单拷贝。
3.转基因水稻植株northern blot分析表明:与对照相比,转反义OsSusiba2基因及其RNAi干扰的水稻株系内源淀粉分支酶表现出一定程度的抑制。转过表达Susiba2基因水稻株系内源淀粉分支酶表现出一定程度增强。
4.糖诱导下转基因水稻的表达分析表明:在蔗糖和甘露醇处理下,野生型、转反义OsSusiba2基因水稻株系和转RNAi干扰OsSusiba2基因水稻株系叶片的内源淀粉分支酶表达基本没有什么变化外,转过表达大麦Susiba2基因水稻株系的蔗糖处理要比甘露醇处理的内源淀粉分支酶表达有所提高,说明Susiba2基因和OsSusiba2基因均受蔗糖诱导表达,而不受甘露醇的影响。
5.转基因稻米直链淀粉含量分析表明:对照和转过表达Susiba2基因水稻的直链淀粉含量变化不大,转反义OsSusiba2基因及其RNAi干扰OsSusiba2基因水稻株系的直链淀粉含量均有明显下降的趋势,特别是OsSusiba2基因反义RNA转基因水稻株系SRR11和OsSusiba2基因RNAi干扰转基因水稻株系SRI86降幅较大。
6.转基因水稻植株表型分析表明:对照和转过表达Susiba2基因水稻在表型上基本接近;转RNAi干扰OsSusiba2基因水稻株系和前两种相比无效穗数增多,而引起结实率、实粒数等下降;转反义OsSusiba2基因水稻株系则下降更为明显。
7.转基因水稻的产量性状分析表明:转反义OsSusiba2基因及RNAi干扰OsSusiba2基因水稻株系比对照及过表达Susiba2基因水稻株系的单株瘪粒数明显增多,而单株实粒数和单株实粒重则明显减少,千粒重则无明显变化;转过表达Susiba2基因水稻株系和对照相比无较大变化;转反义OsSusiba2基因及RNAi干扰OsSusiba2基因水稻株系比过表达Susiba2基因水稻株系的单株穗数有明显的增多,而结实率和穗实粒数则明显减少。
8.转基因水稻碳水化合物分配分析表明:转过表达Susiba2基因水稻根茎叶干重比较对照、转RNAi干扰OsSusiba2基因水稻株系和转反义OsSusiba2基因水稻株系均有所提高,说明转过表达Susiba2基因水稻可表现为更高的生物学产量。
Rice is an important food crop in china.With the completion of rice genome sequencing,and it has been as an important monocotyledonous model species of molecular genetics studies for relate small genome and high efficient transformation system.
Formation of rice production and quality is a very complex physiological processes. Food for people is endosperm, which about 90% of milled rice, endosperm in rice directly influences on the yield and quality. so studying on genes of rice endosperm-related development will have great significance both basic theory and practical application.
Firstly, In this study, we will build rice promoter trap system by T-DAN tags, Obtaining 25,000 rice promoter trap lines, 63 of rice promoter trap lines were expressed by screening GUS expression from 2,653 lines, Including 24 endosperm-related lines, and preliminary function analysis of a rice promoter trap line of endosperm-related development, It will provide material and theoretical basis for studying on endosperm-related development gene by promoter trap system of T-DNA tagging.
Secondly, Transcription factor Susiba2 (Sugar signaling in barley) related to barley endosperm development and rice OsSusiba2 gene were cloned based on transcription factor spf1 sequences, and it has 80% homology between Susiba2 and OsSusiba2. In this study, it will construct antisense RNA vector of OsSusiba2 gene, RNAi vector of OsSusiba2 gene and overexpression vector of barley Susiba2 respectively.harboring them into rice gemone by Agrobacterium-mediated, Studies on endosperm development and starch metabolism in rice by their offspring.
Main contents and results are as follows:
Part I: Screening rice promoter trap lines of endosperm-related development
1.The binary vector pCAMBIA1300GUSA-Hyg contained the promoterless β-glucuronidase (GUS) was introdued into Oryza sativa L. Minghui 86 by Agrobacterium-mediated transformation, Obtaining 25,000 rice promoter trap lines. Hygromycin phosphotransferase expression analysis of 2,872 rice promoter trap lines of T0 generation showed that 2,653 of them were positive, the positive rate of 92.4%.
2. GUS histochemical assay of 2,653 rice promoter trap lines for two times was carried out in its differential organ at differential stage, including root, stem, leave, spekilet, embryo and emdospern. The data revealed that 63 rice promoter lines were GUS positive ,accounting for 2.73%; 24 rice promoter trap lines were expressed in endosperm, 0.90% of total number tested; nine rice promoter trap lines were expressed GUS activity in embryo, 0.34% of total number tested; 20 rice promoter trap lines were expressed in anther , accounting for 0.70% ; and five, eighteen, seven, five promoter trap lines were expressed in the roots, stem, leaves, hulls respectively.
3.Segregation ratio of hygromycin Phosphotransferase expression revealed that 28 rice promoter lines contained one loci T-DNA inserted from 39 lines, 71.8% among them.Southern blot analysis of the rice promoter trapping lines showed that w9101, w9106 and w9154 are single T-DNA inserted. Obtaining franking sequences of six endosperm-related lines by Tail-PCR and its deTail information of candidate genes .
PartⅡ: Preliminary analysis of rice endosperm-specific promoter trapping line
1.Obtained flanking sequence of endosperm-specific expression promoter trap line w9101 by TAIL-PCR, whom T-DNA was inserted between P0421H01.22 and P0421H01.23 of AP004750 in chromosome 6 by flanking sequence analysis, and P0421H01.23 is the same order with GUS, indicating that GUS activity was expressed by the promoter of P0421H01.23. Bioinformatics analysis shows that P0421H01.23 gene encods a 601 amino acid protein, it may be a hydrophobic peptide transporter, including 12α- helix transmembrane domain like CHL1, which is common characteristics of peptide transporter family, it can be speculated that P0421H01.23 is a peptide transporter gene( temporarily named OsPtr1) . Its promoter has a feature of endosperm-specific expression activity by bioinformatics softwares .
2. RT-PCR analysis showed that OsPtr1was endosperm-specific expression , OsPtr1 was knockouted by T-DNA inseted.OsPtr1-GFP fusion protein was espressed only in plastid membrane by subcellular localization.
3.Full-length cDNA of OsPtr1 was isolated , and overexpression vector Gt1-OsPtr1 was constructed, transgenic rice plantlets of the vector was obtained by Agrobacterium-mediated. OsPtr1 gene has integrated into rice genome by southern blot analysis.
4.Substance content analysis in rice of nitrogen metabolism-related showed that:Protein and amino acid content of promoter trap line w9101 were reduced .Total amino acid ,17 kinds of amino acids of transgenic rice plants of over-expression OsPtr1 have been increased at different levels compared to the control. Arginine among semi-indispensable amino acid ,6 kinds of indispensable amino acid,glutamic acid, aspartic acid, total proteins and amino acids of them are increased obviously.
Part III: Function analysis of Susiba2 and OsSusiba2 genes regulating starch metabolism in rice
1.Constructing vectors of RNAi and antisense RNA of OsSusiba2 gene, over-expression vector of Susiba2 gene. Obtaining T4 generation homozygous of transgenic rice for above three vectors.
2.Southern blot analysis of transgenic rice plantlets showed that two T-DNA were inserted into transgenic lines of over-expression Susiba2(SRB77and SRB)and transgenic line of OsSusiba2 gene for RNAi(SRI90) ; While transgenic lines of OsSusiba2 gene for antisense RNA (SRR11 and SRR39), transgenic line of OsSusiba2 gene for RNAi(SRI86) have only one T-DNA inserted.
3. Northern blot analysis of transgenic rice plantlets showed that: endogenous starch branching enzyme of transgenic rice for RNAi and antisense RNA of OsSusiba2 gene were suppressed and endogenous starch branching enzyme of transgenic rice for over-expression Susiba2 were inproved compared to wt .
4. Northern blot analysis of transgenic rice induced in sugar showed that wild type, transgenic rice of antisense and RNAi OsSusiba2 gene,of which endogenous starch branching enzyme of rice leaves dipped into sugar and manitol were unchanged.but endogenous starch branching enzyme of rice leaves of over-expression Susiba2 dipped in sugar were increased ,compare to in manitol. These indicated that OsSusiba2 and Susiba2 were induced in sugar, and not induced in manitol.
5. Amylose content of transgenic rice analysis showed that amylose content of transgenic rice of antisense and RNAi OsSusiba2 were decreased significantly compare to wt and transgenic rice of over-expression susiba2, Transgenic lines of OsSusiba2 gene for antisense RNA(SRR11) and transgenic line of OsSusiba2 gene for RNAi (SRI86) were decreased most apparently ,while amylose content of wt and transgenic rice of over-expression susiba2 is like .
6. Phenotype analysis of transgenic plantlets showed that spikes number per transgenic plantlets of antisense and RNAi OsSusiba2 were increased significantly compare to wt and transgenic rice of over-expression susiba2. while setting rate , grain filled number of them were decreased apparently. but these phenotypes of wt and transgenic rice of over-expression susiba2 were close on the phenotype.
7. Yield characterics of transgenic rice analysis showed that empty grains and panicles per transgenic plantlets of antisense and RNAi OsSusiba2 gene were increased significantly compare to wt and transgenic rice of over-expression susiba2, and grains filled number, weight of grain filled , seed setting rate, grain filled number per panicle of them is reduced significantly,1000 grain weight of them did not change significantly.
8. Distribution of carbohydrate of transgenic rice analysis shows that dry weight of roots, stems,leaves of transgenic rice of over-expression susiba2 were improved compare to wt, transgenic rice plantlets of antisense and RNAi OsSusiba2 gene , indicating that biological yield of transgenic rice of over-expression susiba2 were improved.
引文
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