Genetic Transformation And Analysis Of Wide Compatibility Gene S5-n And Analyzing Aspartic Protease Gene Family In Rice

Rice is one of the important food crops in the world,so one of the important issues is how to improve rice production.F₁ hybrids between indica and japonica usually demonstrate very strong hybrid vigor,but it is the partial sterility or full sterility frequently observed in most indica-japonica crosses.So this is a major difficulty encountered in the development of such inter-subspecific hybrids.However,a special group of rice germplasm,referred to as wide compatibility varieties,is able to produce highly fertile hybrids when crossed to both indica and japonica.The purpose of this study is to clone a gene S5 which is a major locus for indica-japonica hybrid sterility and wide compatibility,and was previously located on chromosome 6.Here,we show that S5 encodes an AP,which conditioned embryo-sac sterility in indica-japonica hybrids.Our researches have been done based on the previously work according to S5 loci,and it included three ORFs(ORF3-5). Transformation constructs were prepared for ORF3,ORF4 and ORF5 with indica genomic DNA fragments,and they were introduced into the japonica variety Balilla. Examination of the spikelet fertility of the T₀ plants under natural field conditions showed that there was not a statistically significant difference between the transgene-positive and -negative plants of ORF3 or ORF4,indicating that neither of them is a likely candidate for S5.Then,we examed the spikelet fertility,embryo-sac fertility and pollen fertility of transgene-positive and -negative plants of ORF5 construct.It showed that the average spikelet fertility of the positive plants was 2.8%and the average embryo-sac fertility of the positive plants was 12.8%,but the average spikelet fertility of the negative plants was 53.3%and the average embryo-sac fertility of the negative plants was 76.7%.However, the average pollen fertility of the positive and the negative plants were more than 80%. This result showed the low spikelet fertility of transgene-positive plants because of embryo-sac abortion,strongly suggesting ORF5 to be the candidate for S5.Analysis the fertility of T₁ plants,we found the average embryo-sac fertility of ORF5 T₁(B10) transgenic positive and negative plants were 1.3%and 92.3%respectively,and the average pollen fertility of ORF5 T₁(B10) transgenic positive and negative plants were 90.4%and 87.9%respectively,and the average spikelet fertility of ORF5 T₁(B10) transgenic positive and negative plants were 0 and 89.4%respectively,so the results were perfect cosegregation and ORF5 was the S5 gene.To gain insight into the function of S5, the ORF5-NJ11 construct was introduced to 02428,and T₁ progenies were test-crossed with Nanjing 11 and Balilla,were examined for embryo-sac fertility and spikelet fertility. The average embryo-sac and spikelet fertility of C04T1×Balilla transgenic positive plants were 53.6%and 53.5%respectively,and the average embryo-sac and spikelet fertility of C04T1×Balilla transgenic negative plants were 81.9%and 72.8%respectively. The average embryo-sac and spikelet fertility of C04T1×Nanjing 11 transgenic positive plants were 85%and 67.5%respectively,and the average embryo-sac and spikelet fertility of C04T1×Balilla transgenic negative plants were 85%and 63.5%respectively. This results indicate that S5-n is a nonfunctional allele.RNA in situ hybridization showed S5 has a localized expression in the ovule tissues including necellus,integument, megasporocytes and megaspores.It had no detection in leaves and roots.Another,we analyzes the distribution and expression of all genes belonged to the aspartic protease family in rice.We identified 96 OsAP genes by a comprehensive bioinformaitic analysis of rice genome sequence database including TIGR,KOME and NCBI RefSeq.Totally 93 OsAP genes were localized on the 12 rice chromosome pseudomolecules.96 OsAP were divided into three categories in a phylogenetic tree,and the A category was more conserved structure compared to another two categories.The numbers of genes were the largest in the C category,but the gene structure varied much. Expression profile data of OsAP genes in Minghui 63 and Zhenshan 97,using RNA samples from 31 tissues hybridized with Affymetrix rice gene chips were extracted from CREP database(http://crep.ncpgr.cn).Sixty-six of the genes had "present" call in at least one of the stages analyzed.Expression pattern of the sixty-six genes were grouped into three main classes.Classâ… was subdivided into four subclasses.ClassⅠαand classⅠγshowed low expression levels in all tissues analyzed.ClassⅠβand ClassⅠδparticularly showed high expression in root and stamen in one day before flowering respectively, compared to the rest analyzed tissues.Classâ…¡showed low expression in the stages of young panicle development,but they showed relatively high transcript accumulation in vegetative tissues.In contrast,classâ…¢showed high expression level during the stages of young panicle development,but with low expression in most vegetative tissues.To investigate the light regulation of OsAP genes,the microarray data were obtained for the tissues of plumule and radicle treated with light and dark for 48 hr at 48 hr after emergence.A total of 14 OsAP genes were differentially expressed between light and dark treatments in the two tissues in two varieties.For phytohormone treatments,nine OsAP genes in two varieties exhibited differential expression in response to NAA,GA3 and KT treatments.All of 12 pairs of the genes located in the segmentally duplicated regions were found to be represented in the Affymetrix GeneChip,which provided opportunity for comparing their expression patterns.The expression patterns of the two members of seven gene pairs were different in most of the tissues tested,indicating one member of the duplicates might have gained new function.