Unction Analysis On The Beta-1,3-Glucanase Genes Specifically Expressed In Anther Of Arabidopsis

Plant β-1,3-glucanase is pathogenesis-related protein, classified as member of the PR-2family and catalyzed the hydrolysis of β-1,3-glucan (a polymer of β-1,3-linked glucose residues). β-1,3-glucanase can be induced by pathogens or other stimuli, meanwhile, this enzyme may be involved in various physiological and developmental processes, such as cell elongation, cell division, pollen germination and tube growth, fruit ripening, seed germination and dissolution of microspores in tetrads.By SSH method and reverse Northern blot,76differentially expressed unigenes were identified from the plant7365A (male sterile) and7365B (male fertile). Base on the information in Arabidopsis, combining with real-time PCR analysis, both BnA6and BnMSR66were obviously suppressed in expression levels. BnA6and BnMSR66encoded β-1,3-glucanase which might be involved into callose degradation during anther development.In order to know whether the BnA6and BnMSR66directly involved in to callose degradation during anther development, we cloned their homologous genes (At4g14080and At3g237700) in Arabidopsis. At the same time, we cloned the other two β-1,3-glucanase genes (At4g16260and At3g24330). All ORFs of these four genes were transcriptionally fused with the A9, AMS and MYB103promoters to construct the chimeric genes, and they were used to transform into Arabidopsis. As three of four genes had a X8structure which was a class of carbohydrate-binding modules responsible for binding β-1,3-glucan, we cloned the genes lacked the X8structure, and then transcriptionally fused with the A9, AMS and MYB103promoters to construct the chimeric genes which were transformed into Arabidopsis.Through selection and identification on the transformants, we won a total of276transgenic plants of21modified vectors. Among276plants, there were52plants shown male sterility, most of them were from the modified vector using the homology gene of BnMSR66in Arabidopsis removing X8structure. Generally, the transformants with the A9or AMS, or MYB103promoters appeared male sterility.With the further cytological observation for sterile materials, we discovered that there was no significantly difference comparing with wild-type material in the process of meiocyte meiosis forming microspores. With developing the microspores into mature pollen grains, the vacuole inside microspores obviously changed, the microspores could not proceed with normal mitotic divisions to form fertile pollen grains, and the exine (pollen outer wall) of sterile materials was rough compared to the flat exine of wild-type materials.The result of aniline blue staining showed that callose wall of transformants and wild-type material at tetrad period had significant difference. There was almost no callose wall around the microspores at terad stage and little callose deposition in the intervals between the microspores at terad stage in transgenic plants. However, we can detect a strong signal around the whole tetrad in the wild-type materials. Thus, to be sure, the reason lead to male sterility in transformants was that the microsporocyte callose wall was pre-degraded at terad, and the callose pre-degradation directly led to the microspores could not normally develop into fertile mature pollen grains.GUS activity and expression pattern of the target gene by eFP in Arabidopsis revealed that the activation of the promoter was detected in anthers of florets (at the anther developmental stage9,10and11), with no expression in florets at other stage, leaf sheaths, leaf blades, stem and root.Based on the above results, we can conclude that anther specifically expressed beta-1,3-glucanase gene (At3g237700) expressed in advance can cause pre-dissolution of the microsporocyte callose wall at anther development process, which affected the microspores further to develop into mature pollen grains, result in male sterility.