Cloning And Expression Analysis Of Flower-related Genes LFY In Juglans Mandshurica Maxim

Juglans mandshurica Maxim is a deciduous tree in the genus Juglans,family Juglandaceae,which is also an important economic tree species of woody oilseed in China.It's fruit has high economic value,nutritional value and medicinal value,that can have a broad prospect of development and utilization.But because the Juglans mandshurica junior period is long,in natural secondary forest,the number of big diameter wood reaching the fruiting age is less than before and it was difficult to harvest seeds from big diameter wood in the population.The seed quality and seed yield decreased,which severely restricted the development and utilization of Juglans mandshurica.Investigation reveals that there are two kinds of flowering patterns: female antecedents and male antecedents of Juglans mandshurica.The flowering time can no meet because of the the two type,it seriously affected pollination and fruiting of Juglans mandshurica.At present,the molecular mechanism of sexual dimorphism and dichogamy in Juglans mandshurica is unclear.Therefore,this study intends to obtain the LFY gene of Juglans mandshurica by homologous cloning,and the expression of LFY genes in different organs and tissues was quantitatively analyzed by fluorescence.Also,the LFY gene expression vector was constructed and prokaryotic expression was analyzed.In order to lay a research foundation for the molecular mechanism of the flowering of Juglans mandshurica,increase the yield and quality of fruit,promote the development of both fruit and timber forestry industry of Juglans mandshurica.The main results of this study are as follows:1.The cDNA sequence of LFY gene was cloned from the Juglans mandshurica genome by homologous cloning,the length of the sequence encoding region(CDS)is 1158 bp.NCBI Blast alignment showed that: the CDS length of this LFY gene is similar to that other LFY homologous species.Moreover,the homology of LFY homologous gene nucleotide sequences with the same family and the same genus were more than 80%.The cloned LFY homologous gene of Juglans mandshurica were named JmLFY(Gen Bank accession number:KX364241).Take DNA as the template,PCR amplification was performed using the full-length cDNA primers of Juglans mandshurica LFY gene,obtain the JmLFY gene DNA full-length is 3007 bp.The length of DNA sequence from upstream primer to downstream primer was 2868 bp,which contains two introns with the length of 543 bp and 1167 bp,respectively.2.GeNorm,Normfinder and Best Keeper are used to screen and verify the stable reference genes EF1? and Actin2 and EF1? is used as the reference gene for fluorescence quantitative PCR.Fluorescence quantitative PCR results showed that JmLFY gene is always expressed in male and female flower buds and pistil stamens.Moreover,the expression level increased significantly during the transformation from bud to pistil.The expression of JmLFY gene is relatively small in the pistils of pollinated and enlarged fruits.These results indicate that JmLFY gene plays an important regulatory role in flower development.JmLFY gene is also consistently expressed in leaf buds and fruits.The relative expression of JmLFY gene in leaf buds increased during the flowering stage,the expression increment is more than one time of other periods.In the fruit,the relative expression level of JmLFY gene was increased in the last two stages of fruit formation and the expression is more than twice as much as in the previous two periods.Based on the above conclusions,it can be inferred that JmLFY gene is indeed a key gene related to flower formation.3.Using pBM30 rapid cloning kit,after connection,transformation,colony screening,PCR validation and sequencing,successfully constructed JmLFY gene into vector pBM30 and named it pBM30-JmLFY.Transformed the carrier into BL21(DE3)competence cell,the positive bacteria were induced by IPTG and collect the expressed protein products.SDS-PAGE detection of polyacrylamide gel electrophoresis is performed to check the products.The electrophoresis results showed that the expression bands is between 48 KDa and 63 KDa which is consistent with the expected result.It proved that pBM30-JmLFY vector could express protein successfully,and lays a foundation for further study on the protein function of JmLFY gene.