| The middle and lower reaches of Yangtze River are the main producing areas of Brassica napus L.Cytoskeleton plays a key role in sensing environmental stimuli,transmitting stimulus signals,changing dynamic patterns and regulating plant growth and development.Current studies have shown that cytoskeleton plays an important role in plant resistance to stress.However,the screening and functional identification of cytoskeleton-related waterlogging tolerance genes in B.napus under waterlogging stress are rarely reported.In this study,based on the results of RNA-seq in B.napus at seedling stage of waterlogging treatment,qRT-PCR was used for transcriptional analysis and verification,and bioinformatics was used to screen cytoskeleton-related genes in B.napus.Overexpression vectors of important genes were constructed to transform Zhong Shuang6 in B.napus,and functional analysis of transgenic plants was performed.The main results of this study are as follows:(1)qRT-PCR and laser confocal observation showed that cytoskeleton was involved in waterlogging resistance of B.napus from gene levels.qRT-PCR was used to detect the relative transcription levels of 27 cytoskeleton-related genes,and it was found that 27 genes were differentially expressed after waterlogging treatment,and the gene expression was variety-specific,indicating that cytoskeleton responded to waterlogging stress at the gene levels.Among the 27 genes,14 genes belonged to microtubule(MT)skeleton related genes,which were divided into TUA,TUB,MAP65 and MAP70 gene families.Laser confocal microscopy observation showed that the fluorescence intensity of the waterlogging-resistant sensitive B.napus Zhong Shuang6 decreased after waterlogging treatment,while that of the strong waterlogging-resistant B.napus Yang Guang2009 increased.These results indicated that the change of microtubule skeleton-related protein levels by regulating genes expression was one of the mechanisms of response to waterlogging stress in B.napus,and the response mechanism was variety-specific.(2)All BnTUAs have anaerobic-induced response elements except BnTUA3.9,and12 members have abscisic acid response elements.In this study,the BnTUAs gene family was analyzed by bioinfoematics method.The results showed that BnTUAs protein family members can be divided into 3 subfamilies,and 14 protein members are located in the cytoplasm.The evolution of BnTUAs gene family was conserved within species and varied greatly between species.The analysis results of cis-acting elements showed that all BnTUAs gene family members had anaerobic-induced response elements except BnTUA3.9,and 12 members had abscisic acid response elements.The BnTUA2 s subfamily has high transcription levels in all parts of B.napus,suggesting that it may play an important role in the whole growth and development process of B.napus.(3)The BnTUB4 s subfamily may be involved in the regulation of the whole growth and development process of B.napus.All 20 BnTUBs family members have anaerobic induction response elements,which is believed to be involved in the resistance response of B.napus and play an important role in the roots.Bioinformatics analysis was used to identify and analyze 20 members of BnTUBs family.BnTUBs protein family members can be divided into nine subfamilies,and all 20 protein members are located in the nucleus.The BnTUB4 s subfamily is evolutionarily conserved,and its gene structure,motif and conserved domain are similar,which may regulate the same function.BnTUB4 s subfamily members are highly expressed in all parts of B.napus,suggesting that they may be involved in the whole growth and development process of B.napus The results of gene cis-acting element analysis showed that all members had anaerobic-induced response elements,and 18 members had abscisic acid response elements.(4)BnMAP65-1 may be involved in waterlogging resistance of B.napus.and regulated by gibberellin,methyl jasmonate and abscisic acid.28 BnMAP65 s family members were identified by bioinformatics methods.BnMAP65 s protein family members can be divided into 9 subfamilies,of which 14 protein members were located in the cytoplasm,9 in the nucleus,3 in the chloroplast,2 in the endoplasmic reticulum.These results indicated that different BnMAP65 s protein members play roles in different parts of the cell.The gene structure of BnMAP651 s subfamily members is highly conserved,26 BnMAP65 family members all have anaerobic induction response elements,23 members have gibberellin response elements and methyl jasmonate response elements,and 20 members have abacic acid response elements.BnMAP651.1,BnMAP651.3 and BnMAP651.6 were expressed at high levels in roots and leaves,suggesting that BnMAP65-1 might be involved in waterlogging resistance of B.napus and regulated by gibberellin,methyl jasmonate and abscisic acid.(5)Construction and genetic transformation of overexpressed BnMAP65-1 vector.In this study,BnMAP65-1 was specifically amplified from the root c DNA of ZS6 and the line plasmid p MDC43-GFP was obtained by double enzyme digestion of Pac I and Sac I.The promoter p MDC43-BnMAP65-1-GFP overexpression vector was obtained by seamless cloning and recombination technique,sequenced,transformed and screened to obtain positive clones.The overexpressed BnMAP65-1 transgenic ZS6 was constructed by hypocotyl impregnation method,and the positive plants were obtained by resistance screening.(6)Overexpression of BnMAP65-1 improved the waterlogging tolerance of B.napus.Laser confocal imaging showed that BnMAP65-1 was located near the cell membrane and in the cytoplasm.After waterlogging,BnMAP65-1 was mainly located near the cell membrane and its fluorescence intensity decreased,indicating that BnMAP65-1 was mainly co-located with periplasmic microtubules.The results of qRT-PCR showed that waterlogging stress reduced the transcription level of BnMAP65-1,and the MDA content in the Zhong Shuang6 CK group was significantly lower than that in the wild-type control CK group,indicating that overexpression of BnMAP65-1 was beneficial to maintain the stability of cell membrane and reduce the degree of peroxidation of plasma membrane.In addition,compared with the wild-type control,the contents of chlorophyll b and soluble sugar in plants overexpressed with BnMAP65-1 were significantly increased,while the contents of soluble protein were significantly decreased,indicating that overexpression of BnMAP65-1 could enhance the ability of plants to regulate osmotic pressure,reduce the effect of waterlogging stress on photosynthesis,and alleviate the restriction of waterlogging stress on plant growth and development. |
