| As an excellent leguminous forage,Lotus corniculatus has a great utilization prospect,but drought limits its popularization and utilization efficiency.It is an important means to find drought resistance genes and cultivate drought resistant varieties to study the differential expression of genes in plants under drought stress.Suppression subtractive hybridization is an effective method for screening differentially expressed genes.In order to cultivate high drought resistance Lotus corniculatus germplasm,after many years of research,our research group selected high drought resistance Lotus corniculatus germline B08.In order to understand the drought resistance mechanism of B08 Lotus corniculatus germplasm,cultivate high drought resistant Lotus corniculatus and excavate related drought resistance genes.Popularize and utilize B08 Lotus corniculatus in this study,the forward and reverse suppression subtractive library of B08 Lotus corniculatus under drought stress was constructed by suppression subtractive hybrid technique,and 600 positive clones were randomly selected from the forward and reverse library for sequencing analysis.The drought resistance related genes of B08 Lotus corniculatus were screened out,and then the activities of drought resistance related plant hormones,permeability regulators,chlorophyll and antioxidant enzymes regulated by these genes were determined,and the following conclusions were obtained:(1)The B08 Lotus corniculatus SSH cDNA library was successfully constructed.(2)574 and 595 high-quality sequences were obtained from the forward and reverse libraries.The average length of the positive library sequence was 207.5 bp,and the average length of the reverse library sequence was 242.7 bp.Phred was used to assemble 1 169 high-quality sequence data,and 632 Unigene sequences(also known as independent genes)were obtained,of which 178 were forward and 454 were reverse.(3)A total of 632 Unigene were compared with NCBI nt,NCBI nr,Swissport and other databases,and there were 147 genes annotated successfully,and another 485 new genes were found.The results of comparison of 632 Unigene sequences showed that most of these genes were homologous to the genes on chromosome DNA1-6 of Lotus corniculatus genome.(4)Species statistics of NCBI nr annotation showed high homology with Populus tomentosa,apple,spinach,barley and rape.The forward and reverse libraries were classified by GO.The proportion of these genes involved in cell and organelle composition,protein binding,cell metabolic process and catalytic action was larger.(5)The obtained Unigene involves plant hormone synthesis,energy metabolism,photosynthesis,protein metabolism,ion transport,nucleic acid metabolism,signal transduction and so on.(6)Indole-3-acetic acid inducible protein ARG7,Serine/Threonine-protein phosphatase,Calmodulin-like protein,Mitogen-activated protein kinase,etc.It was found to be closely related to drought resistance in plants.(7)The content of proline and soluble sugar in the leaves of the drought-treated group with ARG7 gene expression was significantly higher than that of the control group(P<0.05).On the 3rd,5th,7th day of drought,the contents of proline increased 7.9,8.1,8.4 times,respectively.The drought resistance of Lotus corniculatus was enhanced.(8)The activities of catalase,peroxidase and superoxide dismutase in the leaves of the drought-treated group were significantly increased(P<0.05),and the differential expression of the calmodulin-like protein gene was observed.And the activities of catalase were increased by 4.1 times on the 3rd,5th and 7th day of drought,respectively.1.9 times,4.0 times,peroxidase activity increased 3.7 times,5.4 times,4.6 times,superoxide dismutase activity increased 3.5 times,1.8 times,2.9 times respectively.Improve drought resistance.(9)Under drought treatment,chlorophyll a chlorophyll b and total chlorophyll content in the leaves decreased significantly,but there was no significant change on the 5th day.The chlorophyll a/b values in the leaves of the drought-treated group were significantly higher than those in the control group.And differential expression of calmodulin-like protein gene in treatment group.(10)The levels of IAA and ABA in the leaves of the treated group with serine/threonine protein phosphatase and ARG7 gene expression were significantly increased(P<0.05).On the 3rd,5th,7th day of drought,the content of IAA increased by 1.5 times,1.2 times and 1.3 times,respectively.The content of ABA increased by46.14 %,53.34 % and 46.45 %,respectively,which enhanced the adaptability of Lotus corniculatus to drought environment. |
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