Construction Of B-1,6-glucanase GluM Transgenic Plants And Identification Of Theirresistance To Plant Fungus Pathogens

Plant fungal pathogens threaten the crop production and qualitiy,which are difficult to control by disease-resistant breeding and chemical fungicides.Developing transgenic varieties that resistant to pathogens was an effective strategy in environment-friendly plant disease control.Genetic engineering with special characteristics has been attracted great attention.GluM was a kind of ?-1,6-glucosidic.bond hydrolases,which has been identified from Corallococcus sp.Strain EGB.GluM specificity hydrolyzed ?-1,6-linked glucan,especially for short-chain ?-1,6-glucan.?-1,6-glucan has been described as the link between mannoproteinsand the main ?-1,3-glucan/chitin polysaccharide.Once ?-1,6-glucan is hydrolyzed,the cell wall will be damaged.Based on this case,?-1,6-glucanases have been regarded as an important role in developing durable resistance in crop plants against fungal pathogens.In this study,the model plant Arabidopsis was choosed,and the method.of Agrobacterium-mediated gene transfer was used to construct the transgenic plant.The modified gluM gene fragment and plant expression vector pSuper-1300+ were digested by Hind III and Kpn I to contruct the gluM expression vector.The target gene was introduced into Arabidopsis by Agrobacterium-mediated transformation.The expressions of glum in the transgenic Arabidopsis were identified,54 plants were tested by PCR after screening for acquired tolerance to hygromycin.Based on the PCR analysis,50 plants were verified to be gluM transgenic plants,and we selected part of positive plants and 8 homozygote trangenic plants were obtained in the research.The resistance against Botrytis cinerea suggested that gluM is expressed in the transgenic plants with varying degrees.The plant with superior expression was selected for further research.From the resistance test of transgenic Arabidopsis,Wild type,V and transgenic Arabidopsis were spraying out with spore suspension of B.cinerea.Symptoms were observed after inoculationfor six days,and the lesions were visibly smaller in transgenic plants compared with the wild type.The incidence was also researched for statistic analysis,we found transgenic Arabidopsis(20.32)had lower incidence than WT(44.85)and V(45,93)type.RT-PCR analyses was used to monitor the expression levels of glu M,BCACTIN on Col,V and glum at 0,2,3 dpi respectively.The genes gluMand ACTIN2 were steadily expressed in transgenic plant with the time going by.The RT-PCR data indicated that WT and V plants were more abundant in BCACTIN transcripts,respectively,compared with the transgenic line.Lactophenol trypan blue staining of individual leaves was carried out to detect Botrytis cinerea growth at 4 dpi.Fungal growth was evident in both wild type,V and transgenic plant at 4 dpi with obvious necrotic areas.But the transgenic plants appeared to be sufficient to restrict further fungal growth and disease symptoms while V-type is unable to limited the fungal growth and symptom development.In V-type plants fungal growth increased significantly more than in the transgenic plants.The determination of chlorophyll content showed that transgenic plant with more chlorophyll content was detected compared with WT and V plants.This results indicated thatthe transgenic plant with gluM gene was identified to show resistense against Botrytis cinerea.To identify the antifungal activity of gluMgene in rice,the modified gluM gene fragment and plant expression vector pCAMBIA-1300s were digested by Kpn I and Xba I to contruct the?26-gluMand ER-gluMgene expression vector.The target gene was introduced into rice by Agrobacterium-mediated transformation.To identify the espression levels of the gene ER-gluMin transgenic rice,7 plants were obtained by PCR after screening for acquired tolerance to hygromycin.Pathogenicity of To generation was tested by injection with 1×105spores/ml conidia of M.oryzae P131.After 7 d inoculation,abundant and typical lesions were found on WT and V type leaves compared with the smaller lesions from the Ti generation.The sheath of WT and V and transgenic rice of Ti generation were also inoculated with conidial suspensions from wild-type,M.oryzae P131.Again,few infectious hyphae(0%)were produced on the ER-GluM transgenic rice in contrast to abundant infectious hyphae produced on the wild-type(70%),V(43%).These results are consistent with the conclusion that gluM gene has been identified to involve in increasing the transgenic rice resistense against fungal diseases.