The Mechanisms Of Male Sterility On New CMS Yamian A In Cotton

Cotton is an important economic crop, with significant heterosis. Cytoplasmic male sterility (CMS) is not only the basis in crop heterosis utilization, but also is one of the hot topics in the study of biology, and has important significance to understand the genetic mechanism of plant life activities. To clarify the male sterility mechanism, we research cytoplasmic male sterility line Yamian A and its maintainer line at morphology, cytology, physiological and biochemical, mitochondria RAPD analysis, transcriptome, differences in proteomics, genetics and restoring and maintaining relationship. The main results were as follows:1. Floral morphology showed that:There were distinct differences of floral morphology between sterile line and maintainer line. The anther of sterile line was small, dark brown, no pollen grains,and couldn’t shed pollen,but its pistil was in the normal development; The anther of maintainer line were hypertrophy, cream, spread out was full of the whole anther after cracking. There were significant levels of differences in the petal width and ovary diameter, and the extreme significant levels of differences in the flower fresh weight, petals length, petals long x width, head length, filament length, anther length and width, anther length x width index of the flower organ between sterile line and maintainer line.2. Three cytoplasmic male sterility lines (Yamian A, Harknessii A and JinA) were chosen as materials, the differences of their mtDNA were analyzed by the method of RAPD, the results showed that we have gotten characteristic bands from the amplified results through primer E89397respectively, and these cytoplasmic male sterility lines were different each other owing to the differences of their mtDNA.3. Microspore abortion of Yamian A occurred mainly between the stages of sporogenous cell and leptotene. In the microscopic structure, the indications of abortion were that the sporogenous cells were adhesive, sporogenous cells were without nuclei, then some of them were dissolved at the sporogenous cell stage, the sporogenous cells developed into microsporocyte without going through disintegration, then the nucleus disappeared, and completely disintegrated before the leptotene stage. Compared with the fertile line, the tapetal cell development in the sterile line were delayed, both the tapetal cells and middle layer cells did not collapse in the whole process of anther development. All these abnormal phenomenon resulted in the formation of pollen sacs without pollen grains finally. In the submicroscopic structure, the corresponding tapetal cells also showed an abnormal phenomenon of mitochondrial degradation, while the sporogenous cells and microsporocyte showed degradation signs of mitochondrial cristae, endoplasmic reticulum fracture, and so on.4. The physiological and biochemical studies revealed that enzyme activity and photosynthetic parameters relate to fertility. In terms of enzyme activity, The peroxidase activities in the abortion flower buds of Yamian A were significantly higher than that in its maintainer Yamian B, but its succinodehydrogenase activities were lower than that in its maintainer Yamian B, the superoxide dismutase and cytochrome oxidase activities in the leaves and flower buds of different development stages were both lower than that in its maintainer Yamian B. In terms of photosynthetic parameters, The net photosynthetic rate in the leaves of Yamian A were lower than that in its maintainer Yamian B from squaring to boll-opening stage; The transpiration rate and intercellular CO2concentration in the leaves of Yamian A were lower than that in its maintainer Yamian B in the whole growing period except squaring stage; During the squaring and boll-opening period, the stomatal conductance in the leaves of Yamian A were apparently higher than that in its maintainer Yamian B, whereas the stomatal conductance in flowering and boll period turned out contrary. The water utilization ratio in the leaves of Yamian A were higher than that in its maintainer Yamian B only at flowering stage, and remained quite low in the rest of the time.5. CTAB methods, hot borate method and five different RNA extraction kit come from different biology company were compared in the purity and integrality extracted the total RNA from the bud and leaf of Yamian A. The results showed that RN09and RN37Kit of Aidlab Biotechnologies Co.,Ltd could isolate high-purity and highly-complete total RNA at a higher productivity and were better appropriate for the extraction of total RNAfrom cotton buds than other methods and kits.6. The transcriptome study in the flower buds of before, middle and after microspore abortion stage on the cytoplasmic male sterility lines Yamian A and its maintainer Yamian B by means of cDNA-AFLP. Difference banding exhibited550pieces by polyacrylamide gel electrophoresis with226pieces specific difference banding from in the flower buds of middle microspore abortion stage on the Yamian A, accounting for41.09%of total species. There were differences both in quantity and nature among the difference banding with fifteen important expression types in different development period of bud of sterile line and maintainer line.132TDFs were chosen to recycle, secondary amplify, clone sequencing and got99TDFs nucleotide sequences with98TDFs which could search homologous sequences in the NCBI database. These different segments played a very important role in biological process of the catabolism, biosynthesis, endoplasmic reticulum stress reaction and so on, located in the cellular components of mitochondria, apoplast, chloroplasts and so on, involved in molecular functions of the molecular structure, transcription regulation, antioxidant activity and so on, using the Gene Ontology database. These different segments were involved in the metabolic processes of beta alanine metabolism, RNA degradation, protein transport, carbon metabolism and so on, by the analyzing of KEGG database.7. Seven differentially expressed cDNA fragments from cDNA-AFLP were chosen to verify expression pattern by semi-quantitative RT-PCR and fluorescence quantitative PCR. The study showed that the results of RT-PCR, qRT-PCR and cDNA-AFLP on the differentially expressed cDNA fragments were coincident, and experimental study proved the reliability of this cDNA-AFLP technology and feasibility of this study.8. The cDNA full length Class III peroxidase gene from the cotton cytoplasmic male sterile line Yamian A was cloned by three kinds of technologies of cDNA-AFLP, TAIL-PCR and3’-RACE, and was named GhPrx65.The whole length of this gene was1275bp and encoded a protein composed of334amino acids. The DNA sequence analyses had revealed that GhPrx65had the highest consistency with Class Ⅲ peroxidase of cocoa. GhPrx65had a proximal heme-ligand signature, eight conservative cysteine residues, and three phosphorylation sites of serine, threonine and tyrosine. GhPrx65expression was detectable only in the anther with the stage of sporogenous cell division by semi-quantitative RT-PCR and fluorescence quantitative PCR.9. We optimized the protein extraction method and the two-dimensional electrophoresis conditions, and obtained one serial of two-dimensional electrophoresis system which be suitable to cotton buds. In this system, the protein expression profiles with high definition, high resolution,and pH3-10NL were got when the the total protein of buds in cotton sterile line and maintainer line was extracted with improved phenol method, and the immobilized pH gradient(IPG) strips with24cm, pH3-10NL were used, and800ug protein sample were added in electrophoresis.10. The different-display proteomics analysis in the cytoplasmic male sterility lines and its maintainer by technologies of the optimized two-dimensional electrophoresis, mass spectrometry(MS) and bioinformatics and so on. The two-dimensional electrophoresis map of the buds at two abortive critical period from sterile line and its maintainer line(A2, B2, A3and B3) were respectively detected1013,1110,1112and1127protein spots which were analyzed by PDQuest8.0.1software. There were five differentially expressed proteins between A2and B2, and nine differentially expressed proteins between A3and the B3by difference and statistics analysis(Expression of differences>2times, Stud-t99%), and there were differences both in quantity and nature among these differentially expressed proteins.11differentially-expressed protein spots were chosen and excised for MS identification, and11proteins were identified as significant results by using MASCOT search NCBInr green plant protein database and PEAKS6.0search cotton protein database from cotton genome project of the chinese academy of agricultural sciences institute of cotton, and6protein spots among them were retrieved by two ways together. The differentially-expressed proteins played a very important role in biological process of carbohydrate and energy metabolism, endoplasmic reticulum stress reaction and hydrogen peroxide response, fatty acid beta oxidation and toxin degradation, photosynthesis C3carbon reaction and so on, located in the cellular components of mitochondria, chloroplasts and so on, using the Gene Ontology database. According to enrichment analysis by GO Molecular Function of STRING9.05, the differentially-expressed proteins played a very important role in binding metals. The differentially-expressed proteins involved in the metabolic processes of photosynthetic organisms carbon sequestration and dysplasia dicarboxylic acid metabolism, glycolysis and sugar metabolism, photosynthesis, oxidative phosphorylation process and so on, by the analyzing of KEGG database. The differentially-expressed proteins were mainly involved in interaction of carbohydrate metabolism according to protein interaction relationship analysis by STRING9.05. The mRNA level of these differentially-expressed proteins was studied by using qRT-PCR. The results showed that the differentially-expressed proteins gene of the sterile line and its maintainer line expression were detected in seven stages of the anther development, and the expression peak were mainly detected before the third period.The trend of the expression quantity of the gene and protein were not completely consistent at the mRNA level, such changes may be due to the specificity of the different gene expression.11. Using the cytoplasmic male sterile line Yamian A of cotton as infertility source, four genetic stability cytoplasmic male sterile line YMA1, YMA-1, YMA2, YMA7and their maintainer line YMB1, YMB-1, YMB2, YMB7were breeded by using method of backcross breeding. Restore material10N93R and10N91R which could restore the sterility of Yamian A were breeded by using method of test cross screening. The North Carolina II (NC II) mating design was used to formulate five hybrid combinations. The separation of fertility in F1, F2and BC1from crosses between10cytoplasmic male sterile line and two restore materials10N93R and10N91R of cotton were statistically analysed. The results showed that all hybrids in F2and BC1segregated in fertility with respectively13:3,3:1, and2:2fertile to sterile ratio.