Study On The Function Of A SUSIBA2-like Gene In Rice

The endosperm of cereal crops has important economic values.Starch serves as the primary carbohydrate storage in the cereal endosperm,which is a mixture of two D-glucose homopolymers,amylose and amylopectin.The ratio between these two starch components in a seed endosperm determines the quality of the cereal gain.The biosynthesis and deposition of starch in the endosperm mainly involve four enzymes, i.e.,ADP-glucose pyrophosphorylase(AGP),starch sythase(SS),starch-branching enzyme(SBE)and starch-debranching enzyme(DBE).Among them,SBE and DBE are essential enzymes in amylopectin synthesis.SUSIBA2,a transcription factor for sugar signaling in barley,belongs to the WRKY super family.It can activate a starch-debranching enzyme—isoamylase gene iso1 and a starch-branching enzyme gene sbeâ…¡b by recognizing sugar-responsive elements(SURE)in the promoter regions of these two genes so as to affect starch synthesis in endosperm.In order to understand whether there is a similar starch synthesis regulation mechanism in rice, the studies were conducted on four aspects:1)Both the sense and the anti-sense expression vectors of SUSIBA2 driven by Ubi promoter were constructed and were used to transform rice mediated by A. tumefaciens.The PCR identification of hpt,SUSIBA2 and Ubi promoter among regenerated plantlets verified that 4 T₀ plantlets with sense expression SUSIBA2 gene were obtained.2)According to the SUSIBA2 sequence,a SUSIBA2-like WRKY gene consisting of 6 exons and 5 introns was identified from the rice genome sequence.A fragment of third exon of this gene was cloned by PCR from rice genomic DNA,which was 455 bp in length and contained a conservative WRKY domain.The cloned fragment was used as the interference sequence to construct an RNAi vector.The vector was transferred into rice and 16 hpt-positive To plantlets were obtained with only 8% transformation rate.Transplanted in the field until seed maturation,the T₀ rice appearred extremely abnormal in grain-filling since grain filling percentage in 2 of them were 0 and 11 of them ranged between 0.5%and 28%.Growed the normal T₀ grain until seedling,6 of them indicated that the segregation of hpt gene among their T₁ generations was approximate to 3:1.3)By designing the primers in the flank of encoding region,the cDNA of this SUSIBA2-like gene was cloned from young panicles of rice using RT-PCR approach. The cDNA was 1921bp in length,in which the ORF was 1755bp long,encoding a polypeptide of 584 amino acids with two typical WRKY domains and 81%identity with SUSIBA2.We named the gene as SUSIRI.Southern blotting analysis indicated that there is only one copy of this gene in the rice genome.Northern blotting showed that the expression of this gene in rice panicles decreased gradually during the process from heading to seed maturation,that was obviously different from the expression pattern of SUSIBA2 in barley endosperm.4)It was known that the sbe1 gene of rice expresses specifically in endosperm.In this work,a 849-bp promoter region of sbe1 gene was cloned from rice genomic DNA. By searching the database of plant cis-acting responsive elements(PLACE)with obtained sequence,the 77 possible regulation elements were found along the cloned sbel promoter region containing the W-box,however,there was no SURE.The cloned sbe1 promoter was fused with the cloned SUSIRI to construct the endosperm-specific expression vectors of sense and anti-sense SUSIRI gene.In addition,a constitutive Ubi::SUSIRI expression vector was also constructed.These vectors will facilitate the study of the function of SUSIRI gene through rice transformation.