| Brassica napus L.is one of the most widely planted oil crops in China.It can not only produce edible oil,but also be widely used as animal feed due to the rich protein content in its cake.Silique dehiscence is an important physiological process of plant reproductive development.In the process,mature seeds are released from plants,the survival range of offspring is expanded,and reprudction is realized.With the development of social ecnomy,the demand and quality of rapeseed oil are getting higher.It has become an urgent demand for modern agriculture to improve the production efficiency of rapeseed planting by improving the level of mechanized production.In rape production,the time and degree of silique dehiscence affect its harvest and processing.However,oilseed rape siliques are easy to dehisce,which is not conducive to mechanized harvest.Due to lack of dehiscence-resistant germplasm,an in-depth study on the regulation genes and mechanism of silique dehiscence in oilseed rape can provide reference for the breeding and screening of rape varieties with dehiscence resistant.In this study,Bna A07g12590 D was cloned from Brassics napus L.ZS11,which is homologous to Arabidopsis thaliana hemicellulase gene MANNANASE7(At MAN7)related to silique dehiscence.We named this gene BnMAN7 and carried out a series of research work on it.Firstly,we preliminarily analyze the protein encoded by BnMAN7 through bioinformatics.The results showed that BnMAN7 is a relatively stable hydrophilic protein,belongs to the glycosyl hydrolase superfamily,and has characteristics of secretory protein such as signal peptides;In order to have a more knowledge of the biological function of BnMAN7,the expression patterns of BnMAN7 in different tissues was analyzed by fluorescence quantitative PCR.The results showed that BnMAN7 was highly expressed in tissues where abscission or dehiscence would occur,such as leaves,flowers and silique.These results indicate that BnMAN7 may be involved in these processses.We used the onion bulb inner epidermis cell subcellular localization system to analyze the subcellular localization of BnMAN7 protein,the results showed that BnMAN7 protein was located on the cell wall,indicating it may play a role as a secretory protein,which was consistent with the results of bioinformatics prediction.The enzyme activity of hemicellulase encoded by BnMAN7 was verified,it could catalyze xylan into xylose,which demonstrate the hemicellulase activity of BnMAN7.BnMAN7 RNAi and overexpression transformants was constructed by hypocotyl transformation method,and we detected the expression of BnMAN7 and genes related to silique dehiscence in transformnts by q PCR.The necessary force for silique dehiscence in transgenic plants which inhibit BnMAN7 expression was tested,and the results showed that the necessary force for silique dehiscence in transgenic plants was significantly higher than that of wild type,which indicated that BnMAN7 gene was involved in silique dehiscence.CRISPR/Cas9 transgenic lines of BnMAN7 gene as target were also constructed in this study,wherther their gene editing happened need to be detected.Large-scale mechanized harvesting is an urgent need for the upgrading of oilseed rape industry,but oilseed rape siliques are easy to dehisce,resulting in huge losses during mechanized harvesting.Due to the lack of dehiscence-resistant variety germplasm,it is difficult to cultivate high-yield and dehiscnece-resistant oilseed rape through traditional breeding methods.In this study,the BnMAN7,which is homologous gene of At MAN7,was cloned and analyzed in Brassica napus L..The results suggested that BnMAN7 has hemicellulase activity and participated in the regulation of silique dehiscnece in Brassica napus L..It showed dehiscence-resistant in BnMAN7 knock-down plants.This study provides a new idea for oilseed rape molecular breeding and provides available genetic resources for the cultivation of dehiscence-resistant oilseed rape varieties which are beneficial to mechanized harvest. |
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