The Study Of MS2 On The Development Of Anther In Arabidopsis Thaliana And Brassica Napus

The male sterility has been widely used in cross breeding because of the reduction of manpower,financial and material resources in agricultural production.Therefore,studying the mechanism of anther and pollen development plays an important role in crop genetic breeding and the improvement of agricultural production.Previous studies have shown that AtMS2 could control the development of pollen exine.When pollen exine development was abnormal in its mutants,male sterility was induced.Therefore,we carry out a series of functional studies around MS2 in Brassica napus and Arabidopsis thaliana.The main results of this study are as follows:1.Through protein alignment,it was found that the similarity of MS2 in Arabidopsis and B.napus was 94%,and AtMS2 interference fragment with the similarity of BnMS2 interference fragment was 97.69%.AtMS2 interference vector was transferred into Arabidopsis by Agrobacterium-mediated genetic transformation,and six plants of Ti generation of AtMS2 interference were obtained.2.The molecular identification and phenotypic observation of T2 generation of transgenic Arabidopsis showed obvious differences in the flower organs of between transgenic Arabidopsis and wild type.The flower of the non-transgenic Arabidopsis are plump,while the transgenic Arabidopsis are smaller and atrophy,the filaments are short,the pistil is shorter,the anthers are shriveled and the anthers have no obvious pollen adhesion on the surface.Other flower organs with no obvious difference between transgenic Arabidopsis and the wild type.It was found that the surface of pollen in wild was smooth,with an obvious pollen extine,while the transgenic Arabidopsis had completely irregular pollen,the pollen grains were crinkled,the surface had obvious grooves and no obvious pollen extine.It was found that the seeds of non-transgenic Arabidopsis were plump,oval and neatly arranged in the silique.The transgenic A.thaliana did not produce seeds or appeared shriveled seeds and were scattered in the silique.It is preliminarily speculated that interfering of MS2 in Arabidopsis may cause abnormal pollen exine,resulting male sterility in A.thalina.3.Further to the T2 generation of transgenic A.thaliana bud at different periods of cytological analysis,we found that the T2 generation of transgenic Arabidopsis anther tapetum began to degrade during pollen mother cells stage,and the meiosis was abnormal,which could not form normal pollen and lead to anther abortion.4.The expression of BnMS2 in the roots,stems,leaves,flowers,and different developmental stages was analyzed.It was found that the expression of BnMS2 in each flower organs of B.napus was different.Expression of BnMS2 in seedling roots,stems,leaves and flowers is extremely low,while the highest in the bud.5.The BnMS2 protein was expressed in vitro.Western blot showed that the protein size of BnMS2 was between 58 kDa-80 kDa.6.Using the tobacco epidermal transient expression system to analyze the subcellular localization of BnMS2 protein,we found that BnMS2 located in membrane and nucleus.7.Using Agrobacterium-mediated genetic transformation,the BnMS2 interference vector was transferred into the B.napus "Y9",and the molecular identification and phenotypic identification of the transgenic plants of the T0 generation were identified.By observing the morphology of the flower organs,it was found that the stamen of "Y9" was normal,while the stamen of transgenic B.napus is shorter than "Y9" and the anther is smaller than "Y9",other floral organ has no obvious difference.It was found that the pollen grains of "Y9" were circular,and the pollen exine was smooth,with bacula and tectum.The pollen exine was rough,the bacula and tectum were blurred in transgenic B.napus.Compared with the "Y9" pollen,light staining of pollen in transgenic B.napus explained its pollen activity is lower.It is preliminarily speculated that BnMS2 can regulate the development of pollen exine of rapeseed.8.Cytological analysis of RNAi-BnMS2 bud in different stages in B.napus showed that the tapetum degradation is delayed.To sum up,this study carried out the molecular identification and phenotypic identification of MS2 in transgenic B.napus and A.thaliana,preliminarily speculated that BnMS2 may be involved in the regulation of the development of pollen extine.BnMS2 is located in membrane and nucleus.Interference with BnMS2 in B.napus can reduce pollen activity and cause abnormal pollen extine,indicating that MS2 is involved in regulating the development of pollen exine.