Transcriptome Analysis And Functional Identification Of Related Genes In Cold Tolerance Germplasm Of Sweet Pepper Under Low Temperature Stress

Sweet pepper is a non-cold domesticated plant,which is vulnerable to low temperature damage at all stages of growth and development.Low temperature stress is an important factor affecting the yield and quality of sweet pepper in northern China.There is a low temperature response pathway in sweet pepper,and the molecular mechanism of response to low temperature may be different from that of cold domesticated plants.The study of the molecular mechanism of response to low temperature stress in sweet pepper has important practical application value for cultivating new varieties of cold resistant sweet pepper.In the selection of sweet pepper materials,wild sweet pepper has advantages over cultivated sweet pepper in cold resistance,the discovery of the dominant cold resistance gene has an important role,the wild sweet pepper provides a good research material.Many studies have been conducted on the differences of physiological and biochemical indexes between wild and cultivated sweet pepper under low temperature stress,while further studies are needed on the molecular level of sweet pepper under low temperature stress.In this study,wild pepper CA157,cultivated pepper CA52 and introgated pepper CL122 population were used as materials.First,a cold resistance identification system was established to detect the differences in physiological and biochemical indexes of the three kinds of materials.To investigate the molecular mechanism of pepper,cold resistance,the use of gene chip technology analysis compared the cold sweet pepper and cold sensitive pepper under low temperature stress of the transcriptome level,according to the results of the microarray analysis combined with screening differentially expressed genes cloned CaHsfA4a pepper cold resistance related gene,and the CaHsfA4a in sweet pepper the molecular mechanism of cold resistance effect were studied.The main research results are as follows:1.Identification of Chromosomal Region with Cold Resistance in Introgression Sweet Pepper The cold resistance identification system of sweet pepper was established by comparing the difference of growth phenotype and physiological and biochemical indexes between wild pepper CA157 and cultivated pepper CA52 under low temperature stress.The cold resistance phenotype identification of introgmental line CL122 seedlings was carried out by using this identification system.It was found that the leaf growth shrinkage degree after low temperature treatment was lighter than that of cultivated pepper CA52.These results suggested that the genes related to cold resistance might exist in the chromosomal segments of wild sweet pepper.Under low temperature stress,cell membrane damage of CA52,CA157 and CL122 was less than that of cultivated sweet pepper.The survival rate of CA157 and CL122 was significantly higher than that of CA52after 10 days of low temperature treatment and another week of recovery.In the process of low temperature treatment and recovery of the three kinds of sweet pepper materials,the phenotype of low temperature tolerance of osmotic line CL122 was similar to that of wild pepper CA157,and the cold resistance of osmotic line CL122 was obviously higher than that of common cultivated pepper CA52 and other osmotic lines.The cold resistance of CL122 was derived from the chromosomal segment with cold resistance in wild pepper,suggesting that there may be a key gene for cold resistance in the chromosomal segment of wild pepper.2.Analysis of response genes and biological process of resistant sweet pepper under low temperature stress:The gene expression differences of CA157,CA52 and CL122seedlings under low temperature stress were analyzed by gene chip.After 3 days of low temperature stress treatment,125 low temperature responsive genes were detected in CA157 and CL122,and 165 low temperature responsive genes were detected in CA52,suggesting that the differences of these low temperature responsive genes played a role in the cold resistance and sensitivity of resistant sweet pepper.Enrichment of biological processes and functions of clustering analysis showed that there are many in the signal transduction in resistance of two kinds of sweet pepper and biosynthesis,the difference of the higher expression in biological processes such as gene in regulating calcium signal,hormone,ROS process,most of the genes involved will be induced to express,balance of the whole stability of internal environment,It can be seen that the signaling molecules Ca²⁺,hormones and ROS play an important role in the regulation of low temperature in plants,and the expression of related genes in the cold-sensitive material CA52 will be down-regulated,thus affecting the biological process of the whole low temperature response of resistant sweet pepper.There is a difference,which makes the cold resistance of CA157 and CL122 significantly higher than that of CA52 under low temperature stress.These results indicate that cold tolerance genes play an important role in regulating the molecular mechanism of low temperature.Under low temperature stress,CBF（C-repeat Binding Factors）pathway,phospholipid biofilm regulation pathway,Calciumion Calmodulin(Ca^{2+_</sup>Ca M)pathway,Calciumion Calmodulin（Calciumion Calmodulin）pathway,Calciumion Calmodulin（Calciumion Calmodulin）pathway.Seven metabolic pathways,such as Reactive Oxygen Species（ROS）pathway,plant hormone regulation pathway and plant energy metabolism pathway,were significantly adjusted and differed under the response of resistant materials.3.Analysis of the mechanism of heat shock transcription factor CaHsfA4a regulating cold tolerance of sweet pepper:Cas Hsp22.6,a sweet pepper small heat shock protein gene with significantly different expression levels in response to low temperature stress,was screened and cloned in the early stage.Studies confirmed that Cas Hsp22.6 expression was induced by abiotic stress such as low temperature.Then,by screening the transcription factor library,A heat shock transcription factor CaHsfA4a,which was expressed in the upstream of Cas Hsp22.6 under low temperature stress,was identified.The molecular regulation of CaHsfA4a gene on cold tolerance of sweet pepper was studied from multiple levels of transcription regulation and protein interaction,with the A4 type heat shock transcription factor CaHsfA4a as the starting point.The expression level of CaHsfA4a in sweet pepper under low temperature stress was detected by q RT-PCR and Western Blot hybridization.The spatiotemporal specific expression pattern analysis showed that the expression level of CaHsfA4a in sweet pepper was positively correlated with low temperature induction.The expression pattern and function of the cloned CaHsfA4a gene were analyzed.Firstly,the recombinant expression vector p CAMBAI-CaHsfA4a was constructed and transferred into the cotyledon node of sweet pepper by Agrobacterium tumefacien for genetic transformation.The transgenic plants were successfully screened and obtained.Then the transgenic pepper plants were detected,and the results showed that the target gene had been integrated into part of the sweet pepper genome,and the transgenic pepper materials were obtained successfully.The sweet pepper material was treated at a low temperature of 4°C to detect the cold-tolerance related physiological indicators of transgenic sweet pepper,including low temperature treatment at seedling stage and water loss rate of in vitro leaves.The cold-tolerance phenotype of the plant was observed and the survival rate was counted.It was detected that the relative electrical conductivity of the leaves in vitro of the transgenic plant was lower than that of the ordinary plant,and the biofilm stability was better.The growth status of transgenic plants was better than that of normal plants in the 4°C environment,which indicated that the growth of transgenic plants was less affected by the low temperature environment.The results showed that the CaHsfA4a gene was related to the low temperature stress resistance of sweet pepper.The main research results were as follows:the establishment of cold resistance identification system to screen out the introgge system,and the cultivation of’Zhong 30’sweet pepper lines.Furthermore,the differences of physiological phenotypes and transcriptome genomes of the three kinds of sweet pepper were further analyzed,and it was identified that CaHsfA4a could interact with Ca Hsp22.6 in the Hsfs gene family.The results showed that the expression level of CaHsfA4a would increase under low temperature stress,and the cold tolerance of transgenic sweet pepper would be improved.The biological function of cold tolerance of sweet pepper was related to the low temperature induction of CaHsfA4a.}