Cloning And Characterization Of Cinnamoyl CoA Reductase Gene And Analysis Of Their Relationship With Spicy From Capsicum Chinense Jacquin

Pepper is popular all over the world for its unique spicy,high nutritional quality and rich variety of colors.Capsaicinoids were the main source of spiciness.At present,capsaicinoids are widely used in food,military,medical,plant protection and other industries.There is a growing demand for peppers,however,the varieties of high capsaicin,especially those with high capsaicin content,such as ghost pepper,Shuanshuanla pepper and Huangdenglong pepper are limited by the environmental constraints of the place and the negative correlation between the spicy and fruit size.The correlation makes it difficult to improve capsaicin by means of introduction,cross breeding.Therefore,the biosynthesis pathway and regulation of capsaicin has become a research hotspot in genetic engineering to increase capsaicin content.Cinnamoyl-CoA reductase(CCR)is a key enzyme that regulates the lignin synthesis pathway of phenylpropanoid metabolism.CCR activity has an important effect on the spicy of pepper,it can catalyze feruloyl CoA,sinapoyl CoA and p-coumaroyl CoA,which are also precursor substances in the synthesis of capsaicin.In this study,two CcCCRs genes were cloned by PCR and RACE technology and their amino acid sequence structure and evolutionary characteristics were analyzed.The recombinant CcCCR1 protein was obtained by heterologous expression system,and the kinetic characteristics of CcCCR1 enzymatic reaction were further analyzed.At the same time,dynamic analysis of the relationship between CcCCRs and spicy is through the relationship between the content of capsaicin,lignin and flavonoids in the different tissues and growth stages of Huangdenglong pepper,Zhongtian F1 and Haijiao 218 were analyzed,and the qPCR analysis of gene relative expression in the growth stages of CcCCR1 and CCR2 in different tissues.The main results were included as follows:(1)The full length of CCRs were obtained from the stem tip of Huangdenglong pepper by combining the technology of RACE and RT-PCR,Then the characteristic of sequence was analyzed by online websites and software such as DNAMAN,DNAStar.The results revealed that the full-length cDNA sequences of CcCCR1 and CcCCR1 of Huangdenglong pepper were 1362 bp,1423 bp,each containing a complete ORP of 840 bp and 999 bp,which encodes 279 and 332 amino acids respectively.The CcCCRs encoding protein were not secreted protein without transmembrane area or signal peptides and lied in cytoplasm mainly.The sequence alignment showed that the amino acid sequence of CcCCRs had 88.37%homology with the sequences of CCRs in Solanum lycopersicum,Petunia hybrida,contained a typical conserved amino acid residues KNWYCYGK and a NADPH binding sequence.It was found that the CcCCRs protein had closely relationship with Solanum lycopersicum,Solanum tuberosum,Nicotiana attenuate and other Solanaceae plants(2)The recombinant protein of CcCCR1 was successfully obtained by heterologous expression system with the purity of 92%.Subsequently,the enzyme kinetic analysis was carried out to obtain the double-reciprocal plot of CcCCR1 catalyzing the substrate feruloyl CoA to y=36.443x+1.3157,Km is 27.6986μM,Vmax is 0.7601 mmolL-1min-1,Kcat is 1.4822 min-1,the catalytic efficiency Kcat/Km is 0.0535μMmin-1;The sinapoyl CoA equation is y=74.29x+2.1255,Km is 34.9518μM,VmaX is 0.4705 mmolL-1min-1,Kcat is 0.9175 min-1,Kcat/Km is 0.0262μMmin-1;p-coumaroyl CoA curve equation is y=43.992x+1.2038,Km is 36.5443μM,Vmax is 0.0443 mmolL-1min-1,Kcat and Kcat/Km were 0.8307 min-1 and 1.6198 μM min-1.Feruloyl CoA showed the highest affinity and p-coumaroyl CoA showed the highest conversion number,but the highest catalytic efficiency and the best substrate was feruloyl CoA.;Further studies showed that the optimal enzymatic hydrolysis temperature at 30℃ and the optimum enzymatic pH hydrolysis was 6.(3)Capsaicin was detected in all tissues and various developmental stages,in which the capsaicin content of the placenta was greater than that of the pericarp,and the accumulation of capsaicin reached the peak during the color change period;the fruit lignin of Huangdenglong pepper was during development,it first showed a downward trend until it 40 day after flowering.At the same time,the corresponding lignin content in stems and leaves of all varieties was also high,but the correlation analysis of all tissues showed that lignin was negatively correlated with capsaicin,the correlation coefficient was-0.233,the correlation coefficient was-0.233,the results indicate that capsaicin and lignin have significant competitive relationship.The flavonoid of Zhongtian F1 and Huangdenglong pepper fruits increased at first and then decreased,and the peak value of it was always earlier than that of capsaicin.The relative expression of CcCCRs gene was analyzed by qPCR.Results showed that the CcCCR1 was the highest in the stem of Huangdenglong pepper and the lowest in the 40d pericarp of Zhongtian F1 and significantly higher in the tissue with low capsaicin than other tissues.The expression of CcCCR1 was the highest in 20 d pulp of Huangdenglong pepper,and the lowest was 40 d pericarp of Zhongtian F1,and the capsaicin content was lower in the tissues with high expression level.Through this study,the expression of CcCCRs gene and the accumulation of capsaicin,lignin and flavonoids were revealed,and the enzymatic properties of CcCCR1.Those provide a theoretical basis for the regulation of capsaicin metabolism pathway.