Analysis Of Transcriptome And Relate Regulatory Factors Of Different Phaseolus Vulgaris Lectin Levels

Common bean is a popular vegetable which are rich in nutrients such as protein,minerals and vitamins,and is one of the main vegetable crops that people like to eat.However,poisoning incidents caused by improper consumption of kidney beans happen occasionally.The main reason is that the high temperature during the cooking process and its duration are not enough to completely destroy the anti-nutritional ingredients such as Phytohemagglutinin contained in the beans.In recent years,related research on Phaseolus vulgaris PHA has become a hot spot,focusing on the structure and function of PHA protein and subunits.However,it is rare to report on the synthesis of Phaseolus vulgaris and the molecular synthesis regulation mechanism at the content level from the germplasm of Phaseolus vulgaris.In this study,kidney bean germplasm with different levels of PHA,pods at different developmental stages after flowering were used as materials,PHA level determination and transcriptome sequencing were carried out.After comparison,candidate regulatory factors were screened and partial functional verification was carried out to explore kidney beans.The pathway of PHA synthesis regulation and reveals the molecular regulation mechanism of the PHA synthesis level of bean pods,which is of reference value for the implementation of bean germplasm innovations with lower or higher levels of PHA.It will help to eradicate the hidden dangers of kidney bean edible toxicity from the source.Provide reference for artificial preparation of Phaseolus vulgaris PHA.The research content and results are as follows:1)Planting and sampling of experimental materials: A total of 12 commercial pods with different levels of PHA content,including the sequencing bean materials SJ-0053(high PHA level)and SJ-0119(low PHA level),were planted and placed in the pods.Samples were taken at different developmental periods(4 d,6 d,8 d,10 d,12 d,14 d and 16 d after flowering)for measuring PHA content and extracting RNA for q PCR verification.2)PHA content detection: SJ-0053 and SJ-0119 two bean pods samples with high and low PHA levels in different periods were extracted with PBS for protein extraction method,and PHA content was measured by rabbit red blood cell coagulation method.The PHA content in the pods of the two materials changes with the development of the pods,that is,the accumulation of PHA in the pods mainly occurs 10 days after flowering,and the PHA content of the pods of SJ-0053 gradually far exceeds that of SJ-0119.Previously,SJ-0119 The PHA content of pods is slightly higher than that of SJ-0053.3)Screening transcription factors: through the analysis of the transcriptome data and PHA content data of the two materials of SJ-0053 and SJ-0119 in the early stage(Here are the pods on 6d,8d,10 d,12d,14 d and 16 d after flowering),combined with the analysis of gene differential expression between samples,and the correlation analysis of gene expression and corresponding PHA content,Screened out 9 transcription factors that may be related to the regulation of PHA expression,and designed 11 pairs of primers together with two internal reference genes for relative quantification.4)Verification of transcription factors: RNA is extracted from pod samples of different materials in different periods,c DNA library construction and q PCR are performed,and the data is analyzed,and the previous screening results are compared to verify the correlation between transcription factors and PHA expression regulation.Three up-regulated transcription factors,namely ERF1 and WRKY22,one down-regulated transcription factor,namely WRKY33,and two transcription factors that are in accordance with the change of PHA content,were preliminarily verified,namely SKP1,CBF3 D and MYC2.In this study,phenotypic study,transcriptome sequencing,transcriptional regulatory factor screening and q PCR verification were carried out on the PHA content traits of kidney bean pods.The changes of PHA content during the development of bean pods with different PHA levels were preliminarily explored.Preliminary verification of 6 transcription factors that may be involved in PHA synthesis,of which 3 are up-regulated,1 down-regulated,and 2may be involved in feedback regulation.These provide a certain reference value for revealing the genetic law and molecular regulation mechanism of PHA synthesis in kidney bean pods.