Preliminary Study On The Function Of Mitochondrial Gene Orf290 In Rice

In our laboratory,using HL-CMS YTA as material,a 6.7 kb HL-CMS-related mitochondrial sequence HL-sp1 was cloned and identified.According to the sequence prediction,it contained 2 orfs,one of which encodes 290 amino acids,tentatively named orf290.Based on the overexpression and fertility of orf290 transgenic rice plants,the function of mitochondrial gene orf290 was analyzed.The main results are as follows:1.The target gene orf290 was cloned from YTA,and the Rf1b5' coding for the mitochondrial signal peptide was amplified by nested PCR using MH63 genomic DNA as a template.The overexpression vectors,respectively containing mitochondrial signal peptide(35S :: Rf1b5':: orf290)and not containing mitochondrial signal peptide(35S :: orf290),were constructed by using plant binary expression vector pCAMBIA1305.1,and overexpression transgenic positive plants in YTB were obtained by Agrobacterium-mediated genetic transformation.2.By using the YTA as a negative control and YTB as a positive control,the pollen fertility and bag seting rate of transgenic plants were analysed.It is showed that the pollen fertility of the 35S::Rf1b5'::orf290 transgenic plants was below 30%,some of which was completely sterile.The bag setting rate of those plants was about 50%.It is confirmated that the mitochondrial gene orf290 was in relation to cytoplasmic male sterility in rice.3.Using the rice single-copy gene coding for sucrose phosphate synthase(SPS)as an endogenous reference gene and the hygromycin resistance gene(hpt)as a target gene,the copy number of orf290 in transgenic plants was detected by real-time fluorescence quantitative PCR.The results showed that there were two plants with a single-copy of orf290,and the expression levels of transgenic plants were up-regulated with the increased copy number of orf290 except for a few of transgenic plants.4.The DNA and cDNA sequences of orf290 from YTA and 3 L-orf290 lines with different fertility from the backcross progenies of 3 rice varieties of z616,z604 and z597 with YTB were cloned and compared with each other.The results showed that the 3 L-orf290 lines had no sequence difference in orf290.Whether there is any difference in the upstream and downstream sequence of the ORF remains to be studied.