Screening, Cloning And Expression Analysis Of Mitochondrial Genes Related To CMS UG93 In Kenaf

Cytoplasmic male sterility (CMS)is a ubiquitous phenomenon in plants. It is important to elucidate the mechanism of cytoplasmic male sterility for germplasm innovation and utilization of heterosis in crops. Alloplasmic male sterile lines and their maintainer lines were used to research the mechanism of cytoplasmic male sterility by comparing the difference of mtDNA between CMS line and maintainer line in previous studies, which was interferenced easily by the extra genetic information unrelated to CMS because of the great differences beween CMS line and its maintainer line. Cloning and expression of nad3, nad9, nad4L, nad6, nad9, sdh4, cob, atpl, atp4, rps12, rpsl4, rpl1O, rpl16, rpl2, rpl5, ccmb, ccmc and matR were analyzed to study the mechanism of cytoplasmic male sterility in kenaf, using the cytoplasmic male sterility line UG93A, maintainer line UG93B and F1 (UG93A/FuHong992) as the plant materials, in the studies on the mechanism of cytoplasmic male sterility in kenaf, the main conclusions as follows:1. The coding sequence of the 15 genes in the cytoplasmic male sterility line UG93A, maintainer line UG93B and F1 of kenaf were obtained by cloning. There was no difference among them in genomic sequence except single nucleotide polymorphisms. Analyzing the expression of CMS-associated genes through the Real-time PCR, the expression levels of the genes in CMS line were lower than those in maintainer.2. Comparison and analyzing the relationship between mitochondrial gene RNA editing and CMS, the results showed that RNA editing was no significantly difference in CMS line and maintainer line, suggested that cytoplasmic male sterility of kenaf UG93 may be irrelevant to RNA editing.3. The 5’flanking of atp4 was obtained by cloning. When comparing the sequences, a 4 bp deletion, lbp insertion and 4 SNPs were characterized at the 5’-flanking of atp4 in CMS line. There was no size difference of atp4 mRNA in the 3 experimental materials, but the expression level of atp4 reduced significantly in UG93A compared to UG93B and F1 in binucleate pollen stage. In this stage, the expression of atp4 in UG93A was 0.31 folds, and F1 was 1.32 folds compared to UG93B. The activity of mitochondria complex V in UG93A was 0.9 folds compared to UG93B in binucleate pollen stage. All these indicated that atp4 gene may be associated with CMS, and played an important role in energetic metabolism during anther development of kenaf.4. Through comparing the activity of mitochondria complex V and ATP content in the different developmental stages of anther, the results showed that the activity of mitochondria complex V in the CMS line were lower than those in maintainer line and restorer line at mononuclear and binucleate pollen stage. It showed an increasing trend, the activity of mitochondria complex V was lower at mononuclear pollen stage than that in binucleate pollen stage. ATP content was no significantly difference among CMS line, maintainer line and restorer line at mononuclear stage, showed an increasing trend at binucleate pollen stage. The results showed that energy supply shortage at binucleate pollen stage, was related to male sterility.