Functional Verification Of A Mitochondrial Gene Orf182 From Dl-CMS Rice And Preliminary Study On Its Mechanism

Cytoplasmic male sterility(CMS)crops are an ideal material for studying nucleo-cytoplasmic interaction.Further study on the molecular mechanism of cytoplasmic male sterility provides convenience for breeders to further utilize heterosis in crop breeding.A large number of studies have shown that cytoplasmic male sterility is related to the variation of mitochondrial genomes,and abnormal expression of mitochondrial genes causes cytoplasmic male sterility in plants.Dl-CMS rice is a new type of sporophyte CMS rice,which is independent of the wild abortion type and has no pollen.Mitochondrial gene orf182 is cloned from candidate CMS genes of D1 rice.On this basis,this paper uses paraffin section technology to study the anther abortion period and cytological characteristics of D1 type rice.It verifies the function of mitochondrial gene orf182 by using transgenic technology,and the molecular mechanism of mitochondrial orf182 gene leading to CMS is preliminarily studied by using the prokaryotic system.The main results are as follows:(1)The structures of five anther development stages of rice sterile line(DPA)and maintainer line(DPB)are observed by the paraffin sectorial technique.The five anther development stages are the formation stage of microspore mother cells,the meiosis stage of microspore mother cells,the early and middle stage of the mononuclear nucleus,the late stage of the mononuclear nucleus,and the mature stage of the pollen respectively.The results show that the development of microspore mother cells is different from that of microspore mother cells during meiosis.The degradation of tapetum in DPA is delayed compared with DPB.Fluorescence quantitative PCR is used to analyze the expression differences between DPA and DPB of several tapetum developing-related genes including TDR,EAT1,OsAP25,and OsAP37.The results show that there are significant differences in the expression of four tapetum developing-related genes in sterile lines and maintainers.(2)To verify whether orf182 can lead to CMS in D1 rice,we successfully construct the orf182 transgenic vector(pCAMBIA1301-orf182)by fusing the mitochondrial peptide sequence(ATPy)with the sequence of orfl82.Orf182 is transferred into DPA maintainer Yuetai B(YTB)by the agrobacterium-mediated transformation.And we obtain 12 transgenic positive plants.Ten of them show the characteristic of no-pollen abortion.Using the antibody of ORF182,these transgenic positive plants are detected by western-blot.The results show that ORF182 protein is located in mitochondria under the action of the mitochondrial peptide.The above results suggest that the mitochondrial gene orf182 causes the CMS of type D1 rice.(3)Heterologous expression of CMS related genes usually inhibits the growth of E.coli.To study the effect of orf182 expression on the growth of Escherichia coli,we successfully constructed a prokaryotic expression vector of orf182 gene by using pET-28a.The results show that the expression of orf182 inhibits the growth of Escherichia coli,and the expression of orf182 causes the decrease of the respiration rate of Escherichia coli,the explosion of ROS and the decrease of the content of ATP.(4)The enzyme activities of three key enzymes in the oxidative respiration chain are detected in orf182 gene expressing strain:phosphofructokinase(PFK),pyruvate kinase(PK),and citric acid synthase(CS).The results show that compared with the control strain,the PFK enzyme activity of orf182 is decreased,while the enzyme activities of PK and CS are significantly decreased.