| Cytoplasmic male sterility (CMS) is the foundation of cotton heterosis utilization and exploring the mechanism of CMS could make it better be used in the practical production. Jasmonates is a kind of important endogenous plant hormone derived from a-linolenic acid. Jasmonates biosynthesis and signaling processes directly affects the development status of floral organs stamens. In the fatty acid metabolism pathway, co-3 fatty acid desaturase direct catalytic to produce a-linolenic acid. The previous transcriptomics studies show, ω-3 fatty acid desaturase downregulated in flower buds of CMS line Jin A, and all the jasmonate synthesis pathway enzymes are down-regulated. Therefore, we hypothesized that jasmonic acid synthesis blocked during the process of the microspore abortive in the sterile line is one of the causes of CMS line Jin A male sterile. In this study, cotton CMS Jin A, Yamian A and their maintainers are used as materials, the ω-3 fatty acid desaturase gene was cloned respectively from it, By studying the regulation of ω-3 fatty acid desaturase to a-linolenic acid and in microspore development process, determined the content of the buds a-linolenic acid and jasmonic acid, and determined the expression pattern of jasmonic acid biosynthesis key enzyme genes, to investigate the relationship between Jasmonic acid and cotton CMS. The results are as follows:1、Cloning omega-3 fatty acid dehydrogenase (fad3) gene, and sequence analysis was carried out. Using a homologous cloning method, cotton CMS Jin A, Yamian A and their maintainers are used as materials, ω-3 fatty acid desaturase genomic DNA was cloned and length of 2000bp, belonging to ω-3 fatty acid desaturase gene family fad3, composed of eight exons and seven introns, encoding 445 amino acids; cDNA sequence length is 1341bp. The fad3 gene encoding a protein only at the 396th amino acid sites are different between sterile line Jin A and its maintainer line; and has two sites was different between sterile line Yamian A and its maintainers. These differences in amino acid sites on protein primary structure, secondary structure has little effect, this shows that differences of fad3 expression is not caused by genetic structure, different types CMS line have different fad3 gene expression. Phylogenetic analysis showed that cotton Jin A ω-3 fatty acid desaturase gene fad3 cDNA has close genetic relationship with the cocoa. Through sequence alignment discovery, cotton Jin A ω-3 fatty acid desaturase FAD3 contain three conserved histidine-rich domain in the amino acid sequence of the protein, and having high homology with other higher plants. This shows that cotton fad3 gene and protein is relatively conservative.2、 Using the Real-Time PCR to analyzed fad3 gene expression of cotton CMS Jin A, Yamian A and their maintainers. Compared with the maintainer line Jin B, CMS line Jin A fad3 gene relative expression levels downregulation in different periods of bud development; compared with the maintainer line Yamian B, Yamian A fad3 gene expression was significantly higher than the Yamian B during the process of the microspore abortive. Thus suggests that the fad3 gene expression may be associated with cytoplasmic male sterility (CMS), different types of sterile lines fad3 gene expression pattern are different.3、Using ELISA to analyzed the content of ω-3 fatty acid desaturase FAD3 in the various buds developmental stages of each material. The results showed that CMS Jin A ω-3 fatty acid desaturase content were lower than Jin B in the whole process of microspore development; compared with the maintainer line Yamian B, Yamian A ω-3 fatty acid desaturase content is more higher at the early stage and the process of the microspore abortive.The change rule of enzyme content and the expression is consistent, indicating that ω-3 fatty acid desaturase content is regulated by the encoding genes at the level of transcription to a certain extent,4. Determined a-linolenic acid and jasmonic acid content of the various buds developmental stages of each material by chromatography. The results show that during CMS line Jin A a-linolenic acid content is less than its maintainer line Jin B in the early stage and the process of the microspore abortive; both a-linolenic acid content is similar in the late microspore abortion. Throughout the whole bud growth and development, Jin A jasmonic acid content is always less than its maintainer line Jin B. Therefore considered jasmonic acid deficiency is probably one of the cause of jin A CMS. Yamian A a-linolenic acid content is higher than its maintainer line Yamian B in the early stage and the process of the microspore abortive; CMS line with the maintainer line a-linolenic acid content was close in the late abortion. Throughout the whole bud growth and development, CMS Yamian A jasmonic acid content is always higher than the Yamian B. It showed continued high levels of jasmonic acid contents may undermine the balance of hormones, It may be an important reason to induce Yamian A male sterile.5、 The relative expression levels of five key enzyme genes of a-linolenic acid synthesis jasmonate pathway in the various developmental stages buds of each material were analyzed. The results showed that α-dioxygenase, allene oxide synthase, allene oxide cyclase, OPC-8;0 CoA ligase and acyl-CoA oxidase gene in CMS line jin A is less than the expression of its maintainer line Jin B during the process of the microspore abortive. Compared with the maintainer line Yamian B, CMS line Yamian A the five key enzyme in the jasmonic acid synthesis pathway are upregulated during the process of the microspore abortive. This shows that the cytoplasmic male sterility and JA biosynthes is related. Jasmonic acid synthesis pathway is blocked, which may be related to CMS Line Jin A male sterility; and excessive synthesis of jasmonic acid may be one of the causes of the Yamian A male sterile. |
