Transgenic Male Sterile Mustard Plants Induced By TA29-Barnase Transformation And The Functional Analysis And Vectors Construction Of Pea Anther-specific Promoter PsEND1

Male sterile as the most cost-effective approach is widely used for the production of hybrids in rice, corn, canola, and some vegetable crops. Natural male sterility resources with practical breeding value are much scarce. Moreover, infertility resources are disproportion for different crops. Which result in the slow development for the utilization of crop heterosis in some crops. In view of the facts, Breeders hope to solve the problem through the genetic engineering male sterility. Currently, the male sterile line in many plants has been obtained by genetic engineering, and has been used successfully in canola, corn, chicory and other crops. Mustard is one of specialty vegetables in our country, although some progress of mustard breeding by cytoplasmic male sterility have been made in recent years, how to explore and create a novel mustard male sterile material is still one of the important contents of mustard male sterility research. Meanwhile, in order to ensure the stability of genetic engineered male sterile line, a male organ specific promoter with strict temporal and spatial expression is one of the key elements. PsEND1(2.7Kb) is a pea anther-specific promoter that very early express in the anther primordium cells. Subsequently, PsENDl expression is restricted to the epidermis, connective, endothecium and middle layer. Therfore, the PsEND1promoter, different from a tapetum-specific promoter, which can be used to ablate specific cell lines during the first steps of the anther development. It can arrests the microsporogenesis before differentiation of the microspore mother cells and no viable pollen grains are produced. Therefore, the abortion of pollens may be more thorough by the expression of a toxic protein controlled by the PsEND1promoter. However, the functional regions in the promoter have not yet been analyzed. And the long sequence of the promoter will also go against genetic manipulation of male sterility gene. In view of the above problem, the fusion expression vector of Barnase gene driven by the tobacco TA29anther tapetum specific promoter, was imported into "Yu-Feng" mustard by agrobacterium-mediated method. Finally, completely male sterile mustard plants were obtained, which broadened the resources of mustard male sterility. Moreover, according to the distribution of cis-acting elements in the promoter, a series primers were used to do5’end deletion of the PsENDl promoter, and5truncated PsENDl promoter were obtained. Then these promoters were fused with GUS and Barnase gene respectively to generate plant expression vectors. All expression vectors were transferred into agrobacterium EHA105to transform Arabidopsis and tobacco. The main results are as follows:1. Transgenic male sterile mustard plants induced by TA29-Barnase gene transformation(1) TA29-Barnase gene was transformed into "Yu-Feng" mustard by agrobacterium mediated hypocotyl transformation, after several rounds screening using herbicide phosphinothricin (PPT), mustard transgenic plants were obtained.(2) Molecular detection of the TA29-Barnase transgenic mustard plantsThe DNA was extracted from transgenic mustard plants for PCR amplification, a630bp PCR fragment was generated in all five transgenic plants, indicated that the TA29-Barnase gene had been integrated into the mustard plant genome.(3) Floral morphology investigation of transgenic mustard plantsThere is no significant difference in the morphology between the transgenic mustard plants and wild-type mustard plants. Compared with wild-type plants, the transgenic mustard plants had the smaller flowers, petals, shorter and thicker pistils, shorter stamen.The anther of transgenic mustard plants is thiner and shriveled, no pollen sacs and pollen.(4) Anthers development cytological observation of transgenic mustard plantsUsing paraffin section and microscopic technique, we found the tapetum cells in anther of transgenic mustard plants suffered early degradation, no normal pollen grains formed, which resulted in anthers abortion.(5) The seeds of transgenic mustard plantsAll the obtained transgenic plants were crossed with wild-type plants and self-crossed when flowering. The results showed that the pods of self-crossed could not become larger and even suffered shedding, no seeds were obtained. Pollinated with pollens from wild-type plants, compared with wild-type plants, the pods of the transgenic plants were a little short, not full, and fewer seeds. 2. Functional analysis vectors construction of PsENDl promoter(1) The DNA was extracted from pea, then the PsENDl promoter was cloned from the pea DNA by primers PSE1and PSE2. Sequencing analysis showed the PsENDl promoter was correct.(2) Obtained truncated PsEND1promoters by PCR.The PS-2+PS-7; PS-3+PS-7; PS-4+PS-7; PS-5+PS-7; PS-6+PS-7primer pairs were used to amplified truncated PsENDl promoters using full length PsEND1as a template respectively, then the PCR fragments were inserted into PSK and sequenced. These intermediate vectors were sequentially named as pskPS-2, pskPS-3, pskPS-4, pskPS-5, pskPS-6.(3) The truncated PsENDl promoters were fused with Barnase gene to get plant expression vectors.The BN-F+BR-R primer pair and template PCABARABN-10were used to amplify Barnase gene by pfu high fidelity enzyme, the resulted PCR fragment was digested with Ncol and EcoR1to insert into the intermediate vector PBI525. Then the truncated PsENDl promoters in pskPS-2,3,4,5,6were used to replace the35s-35s promoter in above intermediate vector. Finally, all the Barnase gene expression cassettes controlled by different truncated PsENDl promoters were digested by HindⅢ and EcoRI enzymes to inserted into PCABarSN/SCG vector at same sites. These fusion expression vectors were sequentially named as PCABarPS-2Barnase, PCABarPS-3Barnase,PCABarPS-4Barnase, PCABarPS-5Barnase, PCABarPS-6Barnase. These expression vectors were confirmed by PCR and restriction enzyme digestion analysis, and were transferred into Agrobacterium EHA105by liquid nitrogen freeze-thaw method.(4) The truncated PsENDl promoters were fused with GUS gene to get plant expression vectors.The Barnase gene in PCABarPS-2,3,4,5,6Barnase vector was replaced by the GUS gene in PBIIN2-1/GUS vector. These fusion expression vectors were sequentially named as PCABarPS-2Gus, PCABarPS-3Gus, PCABarPS-4Gus, PCABarPS-5Gus, PCABarPS-6Gus. All these expression vectors were confirmed by PCR and restriction enzyme digestion analysis, and were also transferred into Agrobacterium EHA105by liquid nitrogen freeze-thaw method.(5) The transformation of arabidopsis and tobaccoThe PCABarPS-2,3,4,5,6Gus expression vectors were transformed into arabidopsis by agribacterium-mediatede floral-dip method, and tobacco by agrobacterium-mediated leaves disc transformation. The transgenic arabidopsis and tobacco will be obtained later on, which can be used for the PsENDl promoter function analysis.