Studies On The Expressional Stability Of Monooxygenase Transgene In Cotton Genome And The Related Transgenic Male Sterility

Upland cotton(Gossypium hirsutum L.) is an important economic crop,also is an alien species introduced most successfully from America continent in China.Due to the poor natural resources of cotton germplasms and only a few male sterile types available in this country,it is difficult to bring a breakthrough in breeding out new varieties with fine agronomic and economic treats by traditional breeding program.Alternatively,by adopting the technology of genetic transformation,to transfer the genes with the functions of promoting or disrupting the plant development into the genome of cotton cultivars is an efficient way of creating out new varieties and new transgenic male sterile materials.In our lab,via Agrobacterium mediated transformation,three transformation constructs,which contain the gene encoding monooxygenase involved in growth hormone biosynthesis,were transformed into upland cotton genome and were proved to have the transgenes integrated in the genome and to be true transgenic.Furthermore,a transgenic male sterile line induced by the mono transgene was isolated from the T₀ population and maintained to T₅ generation.In this study,the expression at transcription level of the target mono gene and the marker NPTâ…¡gene was analyzed for the T₅ generation of the transgenic lines.The characterization of the transgenic male sterile line was also carried out at the levels of morphology and cytology.The main results were as follows:1.A general method with acidic Chlorophenol-GIT was modified to quickly extract the RNAs of young cotton leaves within two to three hours.The modified method is also suitable for the RNA extraction of different parts of cotton reproductive organs,including pedals,anthers,ovules,pollen grains and young fiber cells.2.92%of total 25 transgenic plants and 90.6%of total 32 transgenic plants of T₅ population had positive signals in the experiment of RNA dot-blotting while separately probed with the PCR fragments of NPTâ…¡gene and mono gene.This indicated that the expression of both transgens at transcription level were still stable in the T₅ generation. The results from the northern blot assay further manifested that the target mono gene and the marker NPTâ…¡gene also stably transcribed in the T₆ transgenic plant genomes.3.The morphology of transgenic male sterile plants obviously changed and appeared in the forms of dwarfish and compact plant shapes,shorter and sparser filaments,thinner and longer stigmata and styles,less and smaller anthers,and even some anthers were shrunk,did not open without pollen grains.4.The pollen grains released from the shriveled anthers of male sterile plants were brown,whereas the ones shed from the anthers of the control plants were light red color, while stained with 1%acetyl carmine.The pollen grains of the former were shrunken, small and poor contents,they were abortive.However,the pollen grains of the later were bigger and full of contents,were viable.5.Reversion of the male sterility to fertility was observed among the male sterile and half male-sterile plants.All 53 half male-sterile plants reversed obviously,and 14 of 17 male sterile plants reversed,the remained three plants always kept sterile.Most reversion of male sterility appeared in the flowers blossomed from the period of the3^rd,Aug.to the9^th,Aug.According to the data of agriculture weather lab in the Hua Zhong Agriculture University in Wuhan,the average temperature before 28 days of blossom was 31.9℃,the highest temperature was 35.7℃,and the lowest one was 28.5℃.the average temperature from the3^rd,July.to the8^th,July.Was 31.87℃.Thus possibly a higher temperature is favorable for the reversion of male sterile sterility to fertility,since the higher temperature affect the meiosis processes of pollen mother cells.6.Microscope observations on the meiosis processes of pollen mother cells showed that the male sterility mainly resulted from the abnormalities of the meiosis,including without meiosis and pollen mother cell degeneration,dyad degeneration,tetrad degeneration,triad formation and degeneration,multi-partite formation,amitosis, asymmetric division,asynchronous division,lagging chromosomes,large quantity of micronuclei and so on.