Cloning And Functional Analysis Of MdCYP716B1-like Gene In Apple

Cytochrome P450 is one of the largest family of enzymes in plants and plays a crucial role in stress response and biosynthesis of plants.So far,there have been relevant reports in rice,arabidopsis,tobacco,black cottonwood and other plants.At present,there are few reports on the function of apple P450,and many functional studies are still unclear.In this study,the coding sequence of cytochrome P450 CYP716B1-like gene was cloned from 'Hanfu' apple,its overexpression and silencing vector were constructed.Apple transgenic plants were obtained through genetic transformation,and many differentially expressed genes were obtained by analyzing transcriptome data.To lay the foundation for revealing the function and mechanism of the CYP716B1-like gene in apple.The main results are as follows:1.The MdCYP716B1-like gene sequence was cloned from 'Hanfu' apple by RT-PCR.Subcellular localization experiments demonstrated that MdCYP716B1-like is localized in the chloroplast.2.In 'Hanfu' diploid apple and autotetraploid apple use qRT-PCR analyze the relative expression of MdCYP716B1-like gene in different organs,and the expression level was the highest in the 'Lengfu' diploid root.The stem,flower and fruit were the second highest,and the expression in leaves was the least.In their autotetraploid,the expression of MdCYP716B1-like gene was the highest in fruit,and the expression of root and stem was basically the same in flower and leaf.The amount of expression is low.3.In the promoter analysis of MdCYP716B1-like gene,it was found that the hormone responsive cis-acting element was detected by qRT-PCR.After detection of ‘GL-3' hormone treatment,the expression of MdCYP716B1-like gene was induced by ABA and MeJA to varying degrees.4.The plant overexpression vector and RNAi vector of MdCYP716B1-like gene were constructed,and the apple 'GL-3' was transformed by Agrobacterium mediated transformation to obtain transgenic plants.Four overexpressed transgenic apple lines were obtained and three RNAi transgenic apple lines were obtained.5.By inoculation with apple anthracnose,the silenced plants were lighter than the control plants but the overexpressing MdCYP716B1-like gene plants were not lighter than the control plants,indicating that the MdCYP716B1-like gene is associated with plant-pathogenic bacteria.6.four pathways related to plant immunity were analyzed by RNA-seq,which were plant-pathogen interaction pathway,MAPK cascade signaling pathway,plant hormone transduction pathway and Triterpenoid biosynthesis pathway.Genes such as JAR1,TIFY,PYL4,SAUR72,NPR1,TGA7,WRKY29,WRKY22,WRKY26,RPM1,PR1,SE3,SQS and OSC4 were was selected from the differential genes involved in these three pathways.The results were verified by qRT-PCR,and the results were consistent with the trend of changes in expression levels in the transcriptome data and it was hypothesized that the MdCYP716B1-like gene is involved in the plant-pathogen interaction pathway.