Studies On Sterility Mechanism Of Upland Cotton (Gossypium Hirsutum Linn) LD6A

CMS(cytoplasmic male sterility)is an important agronomic character,which provides a powerful tool for heterosis utilization.It has been widely used in yield improvement and variety improvement of rice,corn,wheat and other crops.Cotton is an important oil and fiber crop,but its utilization of heterosis is seriously restricted due to the lack of CMS germplasm resources.Therefore,it is of great theoretical and practical significance to develop CMS mechanism research,screening and creating new CMS germplasm resources for Cotton Heterosis utilization.The CMS line LD6 A is a new allohexaploid progeny CMS germplasm resource,developed from the distant cross between Zhongmian 16 and Gossypium stocksii.The female parent of distant hybridization is a highly purified line,Gossypium hirsutum(2n = 52,AADD),and the male parent is a diploid wild cotton,Gossypium stocksii(2n = 26,E1).The triploid branches were obtained by distant hybridization,and then the heterohexaploid was obtained by colchicine doubling.After backcrossing with Zhongmian 16 for many generations,LD6 A and Zhongmian 16(LD6B)were near isogenic lines at the cytoplasmic level.Therefore,a lot of redundant information can be eliminated,which provides a very convenient material for the study of CMS molecular mechanism in cotton.In this study,we compared and analyzed the CMS line LD6 A and its maintainer line LD6 B in cytology,mitochondrial genome,active oxygen metabolism,transcriptome and proteomics.The main conclusions are as follows:1.The development process of microspore in anther of male sterile line and maintainer line was observed by paraffin section.It was found that the anther of LD6 A had been abortion in the tetrad stage,and its abortion characteristics were that it did not produce tetrad microspore,tapetum cells did not degrade during anther development,and the pollen sac finally collapsed into a line.The results showed that the ridge of mitochondria in tapetum cells of LD6 A was fuzzy and the space of membrane was narrow;2.The mitochondrial DNA sequences of CMS lines and maintainers were analyzed by RFLP molecular markers.The results showed that there were 10 polymorphisms in nad9,cox2,atp1,atp4,atp9,ccm B,ccm FN,ccm FN,rps4,rpl10,which indicated that in or near the coding region of these genes had changed at the DNA level,which might be related to CMS in cotton;3.The results of physiological indexes related to ROS metabolism showed that the activity of APX and SOD was lower than that of maintainer line at all stages of anther development;before Anther Abortion,the activity of POD was lower than that of maintainer,but after Anther Abortion,the activity of POD was higher than maintainer line;the content of MDA,the end product of ROS metabolism,was higher than maintainer line at all stages of anther development;4.Through the comparative analysis of transcriptome,a toxic protein gene,Abrin,which can inactivate ribosome and cause mitochondrial apoptosis through endoplasmic reticulum pathway,and some other key differentially expressed genes,such as HATs with BTB / POZ and TAZ transcription factor domains,MDH,WRKY40 and atp8,which are mainly involved in including histone acetylation,TCA cycle,respiratory electron transfer chain and oxidative phosphorylation;they are CMS candidate genes;5.Through proteomic study,the expression of some proteins was analyzed.The results showed that the expression of SOD,POD,APX,were down regulated;Abrin and atp8,the candidate genes of CMS,were up-regulated while HATs were down regulated,and the key enzymes of TCA cycle,MDH,IDH,ME,were down regulated;6.Through the multi-dimensional comparative analysis,the expression correlation of Abrin,HATs,ATP8 and MDH were analyzed,and the results of transcriptome and proteome were verified with each other;the expression of some ROS related enzymes such as SOD,POD and APX were analyzed,and the results of physiological indexes and proteome were verified with each other,and some down-regulated expression enzymes IDH,ME related to TCA were found.7.A hypothesis about the mechanism of CMS was put forward based on all the factors: the up-regulated expression of Abrin inactivates the ribosome of mitochondria,the blocked protein translation results in abnormal mitochondrial structure and narrow membrane space;the down-regulated expression of HATs suppresses the expression of some genes;the decreased activities of MDH,IDH and ME,the key enzymes of TCA cycle,increase ROS accumulation;the decreased activities of SOD,POD and APX result in the decrease of ROS elimination ability;at The up regulation of atp8 results in a large consumption of hydrogen ions in the electron transport chain,and the breakdown of mitochondrial structure and function leads to the failure of tapetum degradation and microspore abortion.