Genetic Verification Of Candidate Genes For CMS In Two Nicotiana Tabacum

Tobacco hybrid seeds are widely used in production,which are mainly produced by using some cytoplasmic male sterile lines of tobacco.The cytoplasm of tobacco cytoplasmic male sterile lines was mainly derived from Nicotiana Suaveolens,which belonged to sua CMS.The long-term use of one type of tobacco sterile cytoplasm tends to result in a weak genetic base,which is at risk.Therefore,it is urgent to dig out new cytoplasmic sources to deal with this problem.At the same time,further studying sua CMS and exploring its abortive mechanism is conducive to better use of sua CMS in production.Firstly,the study carried out cytological studies on the flower buds of CMS lines sua K326 and glu K326,which provided cytological evidence for their sterility mechanism.In addition,expression pattern analysis and genetic verification of candidate genes of sua K326 and glu K326 were carried out to explore the causes of sterility.The research results are as follows:1.Cytological studies on CMS lines sua K326,glu K326 and their corresponding maintainer line of tobacco.The results of scanning electron microscopy showed that the aborted phenotype of sua K326 and glu K326 were partial anther missing and malformation,and the number of stamens was usually less than five.This study also observed the abnormality of mitochondria in the anthers of sua K326 and glu K326 by transmission electron microscopy,showing vacuolation and swelling.2.Verification of male sterile candidate genes of sua K326 and glu K326.Three candidate genes（orf433,orf291 and orf261）of sua K326 were amplified and sequenced in CMS line sua K326 and its corresponding maintainer K326,respectively,and the results showed that three candidate genes were found to be specific to CMS line sua K326.RT-PCR results of orf433,orf291 and orf261 showed that only the bands of orf433 and orf291 were amplified in the c DNA of CMS line sua K326,while orf261 did not.Two candidate genes（orf125a and orf134c）of glu K326 were sequenced in CMS line glu K326 and its corresponding maintainer line K326 showed that orf125a and orf134c in CMS line glu K326 and its corresponding maintainer line K326 were different in base sequences,resulting in amino acid variation.The RT-PCR results showed that orf125a could be amplified only in the c DNA of CMS line glu K326,and orf134c could be amplified in the c DNA of both CMS line glu K326 and the maintainer line K326.3.Analysis of the expression patterns of male sterile candidate genes of sua K326and glu K326.orf433 was highly expressed in the roots and lowly in the leaves of CMS line sua K326;orf291 was highly expressed in the roots and lowly in the flower buds less than 1 mm of CMS line sua K326;orf125a was highly expressed in the stems and lowly in the leaves of CMS line glu K326.The three candidate genes of these two CMS lines materials were expressed in five tissues,including roots,stems,leaves,flower buds at less than 1 mm and flower buds at 1-2 mm,indicating that these three genes were constitutively expressed.4.Verification of transgenic tobacco lines.The positive detection of transgenic seedlings of the vector expressed by the fusion of mitochondrial signal peptide（RF53）driven by UBQ promoter and candidate genes showed that the positive rates of T₀transgenic seedlings of five genes orf433,orf291,orf261,orf125a and orf134c were100%,100%,94%,100%and 100%,respectively.The positive detection of transgenic seedlings of the vector expressed by the fusion of mitochondrial signal peptide（RF53）driven by 35S promoter and candidate genes showed that the positive rates of T₀transgenic seedlings of five genes orf433,orf291,orf261,orf125a and orf134c were100%,100%,96%,93%and 100%,respectively.RT-PCR was used to verify the overexpression of three candidate genes（orf433,orf291 and orf261）of sua K326 in transgenic tobacco plants.The results showed that these three genes were successfully transferred into wild tobacco and expressed.Fluorescence quantitative analysis of two candidate genes（orf125a and orf134c）of glu K326 in transgenic tobacco plants showed that both genes have also been successfully transferred into wild tobacco and expressed.