Cloning And Analysis Of BAHD Acyltransferase Gene Related To Piperine Biosynthesis In Piper Nigrum L.

Pepper(Piper Nigrum L.)is a mysterious and old spice in the world.Pepper is a mysterious and ancient spice crop in the world.It belongs to the genus Piperaceae of the order Piperaceae.It originated in India and is now widely planted in the tropical regions of the world.International FAO 2019 statistics show,Chinese pepper planting area and production are ranked fifth in the world,and there is an important role in Chinese agricultural development.The pepper industry is also helping to have a distinctive fragrance industry in my country’s rural revitalization,serving the country’s " the Belt and Road " strategy.Piperine is one of the most important functional substance and quality trait in piper nigrum.‘Reyin-1’(P.nigrum c.v.Reyin-1,Improved variety of Indonesian large leaf species),the main piper nigrum variety in China.The pepperine content is only 4.80%,and there is still a big difference in the international high-quality peppers in the abuses.The pepperine is low,the quality of peppers is not high,and the molecular breeding technology of pepper is limited,and has become a huge bottleneck that restricts the development of Chinese pepper industry.The project team summarized the pathway of piperine biosynthesis in the previous study,and the direct precursors of piperine synthesis are piperidine and piperamide coenzyme A(Piperoyl-Co A).the piperoyl-Co A as an acyl donor,which is a unit of C6-C3 phenylpropylene compound in a phenylene route,by hydroxylation,fatty acid chain extension and piperidine interaction,a series of reactions synthesized with pepperine biosynthesis.BAHD acyltransferases mediated acyl group transfer reaction is the most critical step in pepperine biosynthesis.The acyl transfer reaction is a bond that connects the polyamine material and phenolic substance metabolism.It is possible to accurately control the pepperine biosynthesis,and it is also a key factor affecting the pepper amide’s diversity.This study is based on the genome sequencing data and transcriptome sequencing data of pepper,this study found that BAHD acyltransferase family genes were widely present in pepper,and performed bioinformatics analysis on them.At the same time,using the roots,stems,leaves,flowers and pepper fruits of different periods after flowering as materials,the gene expression patterns were verified by piperine accumulation pattern analysis,WGCNA analysis,and q RT-PCR.Then the BAHD acyltransferase gene involved in the synthesis of piperine in pepper was screened.The candidate piperine BAHD acyltransferase gene was cloned and expressed.The main findings are as follows:1 、 78 BAHD acyltransferase genes were identified from the pepper genome and transcriptome.By aligning with the 80 identified BAHD family genes from 28 species,it was found that the pepper BAHD family could be divided into 7 categories.In contrast to previous classifications of the plant BAHD family,the BAHD family genes in pepper lack clade II members.The 78 pepper BAHD acyltransferase genes were unevenly distributed in23 pepper chromosomes,and were distributed in tandem repeats on chromosome NO.1.2、Multi-sequence comparison and motif analysis of 78 BAHD acyltransferases,the highly conserved characteristic domains were found(HXXXD,DFGWG and YFGN).The newly discovered pepper BAHD acyltransferases sequence also has a wide range of other relative conservative characteristic domains(YFGN),indicating that the BAHD acyltransferases in pepper has differentiated relationships in evolutionary relationships.The physicochemical properties of the members of the pepper BAHD family are quite different.The number of encoded amino acids ranges from 245 aa to 578 aa and the molecular weight is between 26.37 k Da and 62.72 k Da.Most of the members are acidlabile and highly hydrophilic proteins.3 、 Piperine is dominantly accumulated in pepper fruit,and its content gradually increases with fruit ripening.According to the accumulation characteristics of piperine,the BAHD acyltransferase genes involved in piperine synthesis were further screened through gene expression pattern verification and WGCNA correlation analysis.Identified the piperine synthesis candidate BAHD acyltransferase genes Pn1.3303 、 Pn1.3375 、Pn6.2477 、Pn6.2521 、 Pn11.2248、 Pn11.2279 and Pn12.1813,which were consistent with the accumulation pattern of piperine.4、To identify candidate BAHD acyltransferase genes involved in piperine synthesis,based on the expression patterns of candidate genes in 23 different piperine tissues.Pn1.3303 and Pn1.3375(the encoded amino acid sequences contain complete and unmutated characteristic domains,DFGWG,HXXXD,YFGN),which are consistent with the accumulation pattern of piperine,have the highest expression levels and are located in the edge region of the BAHD gene cluster,were selected and cloned.Among them,the length of the amplified sequence of Pn1.3303 is 1524 bp,including the open reading frame of 1353 bp,and the length of the encoded amino acid is 450 aa;the length of the amplified sequence of Pn1.3375 is 1462 bp,including the length of the open reading frame of 1353 bp,and the length of the encoded amino acid is 450 aa.5、Connect Pn1.3303 and Pn1.3375 to prokaryotic expression p GEX-4T-1 vector by Gateway.The target protein fused to N-terminal and GST tag was successfully induced and expressed.The results provide an important genetic resource for the identification of related BAHD acyltransferases in piperine biosynthesis.