Analysis Of Mechanism Of Relative Gene Responses Of High Temperature And Salt Stresses To Chlorogenic Acid Synthesis Pathway In Jerusalem Artichoke Growth In Tidal Flats

Chlorogenic acid(CGA)is a kind of metabolites produced by the shikimic acid pathway during the aerobic respiration of the plant body.It is widely distributed in plants,and has antibacterial,anti-virus,anti-tumor,anti-oxidation and other biological activities.At present,CGA has been widely used in food,medical care,and health care.Studies have shown that chlorogenic acid is rich in Jerusalem artichoke.As a salt-resistant plant is of great significance for the reasonable development and utilization of coastal beach and seawater.In this paper,the "Nanyu No.1" was the main experimental material,besides,the related genes and their functional analysis were studied systematically during the biosynthesis of CGA in Jerusalem artichoke.The main findings are as follows:1.The content of CGA in different tissues and cultivars of Jerusalem artichoke were analyzed.(1)By comparing the contents of CGAin different tissues of "Nanyu No.1"(roots,stems,leaves,flowers and tubers),we discovered the CGA contents in leaves was highest,more than 9 times for other organizations;(2)Comparison with the different varieties of Jerusalem artichoke in Jiangsu Dafeng and Hainan Sanya,the CGA contents in leaves of"Nanyu No.1" were increased with the increase of growth time and it is basically reached the maximum in October(At this time,the CGA contents of "Nanyu No.1" is highest than other varieties).At the same growth time,the CGA content of leaves was found that:before the growth of Jerusalem artichoke(June,July),the content of CGA in leaves of artichoke in Hainan Sanya was higher than that in Jiangsu Dafeng.In the ripening period of Jerusalem artichoke,the content of CGA in leaves in Jiangsu Dafeng was slightly higher than that in Hainan Sanya.Therefore,it is suggested that the temperature may be the main reason for the content of CGA in leaves of Jerusalem artichoke.We cultivated seedlings of "Nanyu No.1" in a laboratory incubator.The study had shown that the content of CGA in leaves at 30 ? was higher than 25 ?.It is presumed that the appropriate high temperature may promote the biosynthesis of CGA in leaves of Jerusalem artichoke.(3)By comparing the contents of CGA in leaves of planting in Jiangsu Yancheng Xinyang(mild salt),Dafeng(moderate salt)and Shuntai(heavy salt),we found that the CGA contents in leaves of"Nanyu No.1" were increased with the increase of growth time and it is basically reached the maximum in October.In May and June,the content of CGA is heavy salt>moderate salt>mild salt,and in August,September and October,the content of CGA is moderate salt>heavy salt>mild salt.We cultivated seedlings of "Nanyu No.1" in a laboratory incubator,and treated with NaCl.We found that the CGA contents in leaves of "Nanyu No.1" were increased with the increase of growth time and the concentration of NaCl.Therefore,it is suggested that the salt stress promotes the biosynthesis of CGA.In summary,the contents of CGA is highest than other organizations and the latter may play an important role in the high temperature and salt stress of Jerusalem artichoke.2.For the lack of the genome and genetic information of Jerusalem artichoke,we sequenced and analyzed transcripts of leaves of Jerusalem artichoke under 25 ?(RT)and 30 ?(HT).(1)Clean reads of 6 samples are up to 60M.After De novo splicing,accessed 81886 of not less than 300bp unigene.(2)Then comparative analysis was carried out on the unigene,which annotated to the NR database for 41808,SWISSPROT database for 30340,KOG database for 2378,GO for database 2533,KEGG database 10406.Involved 25 KOG functional categories,and there were 1064 unigene of the secondary metabolites biosynthesis,transport and catabolism.(3)Of the 81,886 unigene,2720 were specifically expressed in RT and 2319 were specifically expressed in HT.The highest abundance was RuBisCO small subunit,indicating that it played an extremely important role in photosynthesis of leaves of Jerusalem artichoke.The differentially expressed unigene was 4444,among which 2232 were up-regulated and 2212 were down-regulated.The most significant increase was comp 127014_c0_seq3,and it was I-SST.It was presumably beneficial to promote the synthesis of fructans and improve the ability of Jerusalem artichoke to resist high temperature.(4)In the GO enrichment analysis,we found that 1952 GO terms were annotated,of which 199 GO terms were significantly enriched(FDR<0.01).It included 68 molecular functions,18 cell components and 113 biological processes.In the KEGG enrichment analysis,we found that the differentially expressed unigene were annotated to 272 KEGG pathways,of which 8 KEGG pathways were significantly enriched(FDR<0.01).Starch and sucrose metabolism accounted for the largest proportion of 5.35%,followed by phenylpropanoid biosynthesis was 4.63%.It was speculated that the effect of high temperature stress on carbohydrate metabolism and secondary metabolites is very large.(5)We focused on the related gene annotations and expression of CGA synthesis of transcription group.It was found that there were 14 unigenes were annotated to PAL,and there were significant differences in 2 unigenes;HCT/HQT has 38 unigenes annotations,with 8 unigenes significant differences;C3H had 5 unigenes annotations with 1 Unigene had significant differences;C4H had 5 unigenes annotations;4CL had 10 unigenes annotations;CCoAOMT had 9 unigenes annotations,with no differences unigenes were annotated among C4H,4CL,and CCoAOMT.(7)We randomly selected 11 unigene and verified by real-time quantitative PCR.The results were consistent with the transcription group.3.Cloning and tissue expression of the related gene of the CGA synthesis pathway in Jerusalem artichoke:first,the seven related genes of chlorogenic acid synthesis pathways were obtained by electronic cloning technique.Then,designed primers and amplified to the CDS of the full length by HCT,HQT,CCoAOMTl,and the length of ORF was 1293,1272,786 bp,respectively,encoding 430,423,261 amino acids;only part of the fragment was amplified by PAL1,PAL2,4CL1,C3H1,and the length of ORF was predicted to be 2100,2136,1377 and 1536 bp,respectively,encoding 699,711,459,511 amino acids.Finally,the use of real-time quantitative analysis shows that:PAL1 was the highest expression in the root;PAL2 and C4H were the highest expression in the stem;HCT,4CL1,C3H1,HQT and CCoAOMT1were the highest expression in the leaves.4.The results showed that the expression of related genes of the CGA biosynthesis pathway was analyzed by real-time quantitative under high temperature and salt stresses.(1)The related genes of the CGA synthesis pathway was expressed in the leaves of "Nanyu No.1" in Jiangsu Dafeng,and with the increase of growth time,PAL1,HQT and CCoAOMT1 were increased.The related genes of chlorogenic acid synthesis pathway was expressed in the leaves of "Nanyu No.1",which was planted in Hainan Sanya,but there was no significant correlation.The results showed that the expression of PAL1 and HQT in the leaves of "Nanyu No.1" in Jiangsu Dafeng was higher than that of the plant in Hainan Sanya,Quantitative analysis of the leaves of Jerusalem artichoke seedlings treated by high temperature in the laboratory showed that high temperature promoted the expression of PAL1,PAL2,C4H,HCT,C3H1,HQT,CCoAOMT1,and inhibited the expression of 4CL1.(2)Analyzed related enzyme gene expression of the CGA synthesis pathway of "Nanyu No.1" at different growth stages in different salt fields.The expression of HCT,HQT and CCoAOMT1 increased with the increase of the growth time under salt stress.Quantitative analysis of "Nanyu No.1" leaves of treatment with different concentrations of NaCl solution under laboratory conditions,we found that the related genes of CGA biosynthesis pathway were expressed but there are differences,and with the increase of NaCl concentration inhibited of PALI expression and promote the expression of HQT.The above results show that response to adversity,the eight related genes in the biosynthesis of CGA is different.Both high temperature and salt stresses promoted the expression of HQT,and the expression level was,positively correlated with the content of chlorogenic acid.Therefore,it can be inferred that HQT plays an important role in the CGA synthesis pathway of Jerusalem artichoke.The function of HQT is worthy of further study.The above results will provide a theoretical basis for the study of resistance mechanism of Jerusalem artichoke and the development of chlorogenic acid.