Screening And Function Analysis Of Genes Of Endosperm-Related Development In Rice(Oryza Sativa L.)

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 development1.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 line1.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 rice1.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.