Cloning And Primary Functional Analysis Of Dormant Related Genes OsSDR1 And OsSDR4 In FH7185

Rice is one of the important staple food crops in the world,and its adequate production ensures the world's food security.The dormancy of riee seed is an important agronomic trait,which is related to rice grain and seed quality and also directly related to pre-harvested sprouting resistance of seeds.Therefore it is important to explore the dormancy-related genes and carry out their functional analysis using genetic engineering tools.Based on the preliminary study of rice seed longevity at the National Engineering Laboratory of rice situated at the Fujian Academy of Agricultural Sciences.Two genes associated with seed dormancy were found in collaboration with NCBI:(1)The first dormancy-related gene OsSDR1(Genbank sequence number Os03g0810800)is a xanthoxin dehydrogenase.(2)The second dormancy-related gene OsSDR4(Genbank sequence number 0s07g0585700)which are a transcriptional factors.Their function were preliminarily analyzed.The items and results were as follows.a.The expression level of these two genes was significantly different among FH7185 and its parents(FH653 and YH290)in seed embryo by real-time quantitative PCR(qRT-PCR).b.The coding sequences of the genome regions of those two genes were cloned from FH7185 and its parents(FH653 and YH290).The sequences blast indicated that the coding and genome regions of OsSDR1 among the varieties FH7185 and its parents(FH653 and YH290)were homologous.But both genome and coding regions were found with 9 bases deletion compared to NCBI relevant sequences.The sequences of both coding and genome regions of OsSDR4 were homologous between the varieties FH7185 and FH653.But compared with NCBI relevant sequences,there were 33 bases deletion between the varieties FH7185 and FH653.While the sequences of the coding and genome regions of OsSDR4 were homologous between YH290 and NCBI relevant sequences..c.The vectors of these two genes,including over-expression vectors,genome-editing vectors Crispr/Cas9 system and sub-cellular localization vectors were constructed and transferred into the varieties FH7185 and MH86 mediated with Agorbacterium transformation.The corresponding transgenic plants were obtained.The results indicated that among transgenic plants,there were 53 positive plants among 92 putative transgenic plants,while pCAMBIA1302-OsSDR1,had 38 positive plants among 40 putative transgenic plants.OsSDR1-Cas9,produced 55 positive plants among 97 putative transgenic plants with pCAMBIA1302-OsSDR4,164 positive plants among 222 putative transgenic plants with OsSDR4-Cas9.d.The prokaryotic expression vectors of those two genes were constructed and the corresponding interaction proteins were screened by Yeast two hybrid and GST pull-down experiments.The result indicated that an interaction protein of OsSDR1 was found and its Genbank serial number designated as Os05g0140800.The protein was verified as related to seed dormancy.