Transcriptome Analysis Of Lilium Pumilum And Preliminary Exploration Of The Function Of LpPEX5 And LpPEX7 Genes

In recent years,salinization of land has become more and more serious,and the area of saline-alkali land in China alone has reached 99.13 million hectares.In the saline soils of the northeast,only a few species of lilies can grow,and fine-leaved lilies are one of them.The genes of the PEX family are important genes involved in peroxisome synthesis in plants.It has been pointed out that overexpression of genes in the PEX family will increase the number of peroxisomes.and various enzymes in the peroxisomes It is important to reduce the damage caused by saline-alkali stress.The transcriptome was sequenced after the saline-alkali treatment of Lilium microphylla.and after analyzing the sequencing results of the transcriptome,genes with a greater relationship with saline-alkali were identified,and primers were designed based on the transcriptome sequence.The cDNA was used as a template clone to obtain the LpPEX5 and LpPEX7 gene sequences,respectively,and bioinformatics was performed.The expression pattern of the genes was analyzed by real-time quantitative PCR.The PBI121-LpPEX7 and pMCP1-LpPEX5 plant expression vectors were successfully constructed and transformed into Arabidopsis thaliana by Agrobacterium-mediated method.Arabidopsis thaliana with overexpression of LpPEX5 OE and LpPEX7 OE genes was further analyzed for resistance to stress.At the same time,pQE-LpPEX5 and pGEX-LpPEX7 were constructed and purified successfully,which laid the foundation for further research of LpPEX5 and LpPEX7 genes.The specific research results are as follows:1.By sequencing the transcriptome of Lilium microphylla bulbs,a total of 23.4 Gb of data was obtained,and 115061 unigenes were obtained by assembly.After Blast alignment of Unigene sequences with the Nr,COG,Swissprot.and KEGG databases,a total of 56828 mRNA annotations were obtained.A total of 390 differentially expressed genes were identified,of which 155 genes were up-regulated and 235 genes were down-regulated.Eight genes closely related to saline-alkali stress were selected for qRT-PCR verification,and the reliability of RNA-Seq data was demonstrated by qRT-PCR results.2.The LpPEX5 and LpPEX7 genes of Lily of the Evening Lily were successfully cloned from the bulbs of Lilium microphylla.The length of the LpPEX5 gene was 2268 bp,encoding 755 amino acids,and the length of the LpPEX7 gene was 957 bp,encoding 318 amino acids.Conserved domain analysis showed that the LpPEX5 gene belongs to the TPR superfamily and the LpPEX7 gene belongs to the WD40 superfamily.3.Fluorescent quantitative PCR results showed that LpPEX5 and LpPEX7 genes were more expressed in leaves and seeds.After saline-alkali treatment,the expression levels of LpPEX5 and LpPEX7 genes reached the maximum under NaCl and H2O2 stress for 12 hours,and reached the maximum expression levels under NaHCO3 stress for 24 hours.4.The PBI121-LpPEX7 and pMCP1-LpPEX5 plant expression vectors were constructed,and the recombinant plasmid was transferred into Arabidopsis thaliana through Agrobacterium-mediated method for resistance screening,and Arabidopsis plants with overexpressed LpPEX5 and LpPEX7 genes were successfully obtained.And successfully introduced the LpPEX5 and LpPEX7 gene overexpression vectors into Lilium pumilum.5.The Arabidopsis overexpression of LpPEX7 genes germinated in a saline-alkaline environment compared to wild-type Arabidopsis showed that Arabidopsis overexpressed LpPEX7 genes had stronger salts than wild-type Arabidopsis Alkali resistance.6.pQE-LpPEX5 and pGEX-LpPEX7 were constructed,and the conditions for inducing LpPEX5 and LpPEX7 proteins were explored.The proteins were successfully induced and purified.