High Temperature On Non-heading Chinese Cabbage Seedlings Effects Of Proline Metabolism

Non-heading Chinese cabbage(Brassica campestris ssp.chinensis Makino)is an important vegetable to ensure the implementation of the’vegetable basket’project in China.Because of its preference for coldness,it is easy to encounter high temperature stress in summer in southern China,which seriously restricts the planting and annual supply of non-heading Chinese cabbage.Proline is the most widely distributed osmotic adjustment substance in plants under environmental stress such as temperature and humidity,and its content is often used as an important physiological index of plant stress resistance.Proline metabolism is the main way for plants to respond to stress and affect the content of proline.In this study,the leaves of non-heading Chinese cabbage seedlings were used as materials to explore the effects of high temperature stress on proline metabolism of non-heading Chinese cabbage,in order to provide theoretical support for high-yield and high-quality cultivation and stress resistance index screening of non-heading Chinese cabbage.The main results are as follows:1.Proline synthesis and decomposition pathways in non-heading Chinese cabbage in response to high temperature stressIn order to explore the effect of high temperature stress on proline synthesis and decomposition pathway of non-heading Chinese cabbage,three varieties of non-heading Chinese cabbage seedlings(different heat resistance)were selected and treated at 40°C for different times.The proline metabolism related indexes of seedling leaves were measured.It was found that the total amino acid content,free proline content,P5CS(Δ~1-pyrroline-5-carboxylic acid synthase)and P5CR(Δ~1-pyrroline-5-carboxylic acid reductase)activities of heat-resistant varieties were significantly higher than those of normal temperature and heat-sensitive varieties at the same treatment time.The changes of proline metabolism indexes in different varieties were the same.The total amino acid content,free proline content,P5CS,δ-OAT(ornithine aminotransferase)activity increased significantly,P5CR activity increased first and then stabilized,Pro DH(proline dehydrogenase)activity decreased significantly,and the proline metabolism level of heat-tolerant varieties was high.Under high temperature stress,the free proline content of non-heading Chinese cabbage was positively correlated with P5CS and P5CR activities,and negatively correlated withProDH activity.Proline synthesis and decomposition pathways in non-heading Chinese cabbage respond to high temperature stress,promote proline synthesis and inhibit proline decomposition.2.Identification and expression analysis of proline transport pathwayProT gene in non-heading Chinese cabbageIn order to explore the function ofProT(proline transporter)in the proline metabolic pathway of non-heading Chinese cabbage,the whole genome identification ofProT gene in non-heading Chinese cabbage was carried out.The results showed that the BchProTs contained 6 members,and the gene was located on 4 chromosomes.The amino acid sequence contains a common conserved domain(Aa-trans),with 11-12transmembrane domains,which is a typical transmembrane protein;the BchProTs gene structure contains a small number of introns and the structure is conserved;fragment replication was the main expansion mode of BchProTs,and the Ka/Ks value of collinear gene pairs was less than 1,indicating that BchProTs had undergone purification selection.Collinear analysis and phylogenetic analysis with multiple species showed that non-heading Chinese cabbage had the closest relationship with Chinese cabbage.The BchProTs promoter region contains a variety of abiotic stress and hormone response elements.Public transcriptome data and real-time fluorescence quantitative PCR showed that BchProTs had spatiotemporal expression specificity,and the expression levels in different tissues were different.Under high temperature stress,the expression levels of BchProT1,BchProT4,Bch P5CS1,Bch P5CS2 and Bch P5CR were up-regulated,and the expression level of Bch OAT did not change significantly.The expression of BchProDH was up-regulated under high temperature stress,and the expression of Bch P5CDH was down-regulated.It indicated that BchProT1,BchProT4 and some proline metabolic enzyme genes responded to high temperature stress.3.Cloning,transformation and response to high temperature and drought stress analysis of BchProT1 in non-heading Chinese cabbageIn order to explore the expression location of BchProT1 and its expression pattern in response to high temperature stress,BchProT1 was cloned and a binary composite vector was constructed for subcellular localization experiments.It was found that BchProT1 protein was localized on the plasma membrane,which was consistent with its transport function.BchProT1 was overexpressed in Arabidopsis thaliana by floral dip method,and T3 transgenic Arabidopsis thaliana was obtained after purification.Real-time quantitative PCR analysis showed that the expression level of BchProT1 was significantly higher than that of wild type Arabidopsis thaliana,and BchProT1 was successfully overexpressed in Arabidopsis thaliana.The study of transgenic Arabidopsis showed that there was no significant difference in free proline content between transgenic Arabidopsis and wild-type Arabidopsis under normal growth conditions,and the expression of proline synthase gene and AtProTs in transgenic Arabidopsis was significantly lower than that in wild-type Arabidopsis.The high temperature stress treatment of transgenic Arabidopsis and wild-type Arabidopsis showed that the free proline content of transgenic Arabidopsis leaves was significantly higher than that of wild-type Arabidopsis.Heterologous overexpression proved that BchProT1 is one of the key genes in the proline transport pathway,which is involved in the regulation of the proline transport metabolic pathway and responds to high temperature stress.