Transcriptome Sequencing Of Lonicera Japonica And Cloning And Expression Analysis Of AP1 And AGL19 Gene

Objective: Total RNA extraction method and transcription group of Lonicera macranthoides flowers between Xianglei varieties and conventional varieties were built.MADS-box genes which had differential expression were screened in two varieties.Related genes were cloned and made expression analysis,in order to explore the intrinsic reasons of the special characteristics of Xianglei varieties,such as non-expansion of corollas and long budding period.Methods: 1.SDS method,modified CTAB method,common kit method and polysaccharide polyphenol kit method and modified polysaccharide polyphenol kit method were used to extract total RNA from flowers of L.macranthoides respectively,and to detect their concentrations and purities.2.Solexa high throughput sequencing technology was used to study the transcriptomes of two varieties,to screened out significant differences of expression levels and genes related to flower development in MADS-box gene.3.Through RT-PCR and RACE,cloned the full-length sequences of AP1 and AGL19,and carried out bioinformatics analysis.4.Real-time fluorescence quantitative PCR was used to analyze its expression in different organs and florescence.Results : 1.The most suitable method for RNA extraction from L.macranthoides flowers is the modified polysaccharide polyphenol kit method.2.It was found that there were significant differences of expression levels and genes related to flower development were 40 in MADS-box gene.3.The full-length sequences of AP1 and AGL19 were cloned.Lm-XL-AP1(accession number in Gen Bank: MF139 138)and Lm-AP1(accession number in Gen Bank: MF418 642)both consisted of a 729 bp open reading frame(ORF)encoding a protein that contained 242 amino acids.Lm-XL-AGL19(accession number in Gen Bank: MF426 681)and Lm-AGL19(accession number in Gen Bank: MK419 948)both consisted of a 654 bp open reading frame(ORF)encoding a protein that contained 217 amino acids.4.AP1 was expressed in flowers,stems and leaves of two varieties,but the expression in flowers was significantly higher than that in stems and leaves.Lm-XL-AP1 expression was highest during the sixth flowering stage,while Lm-AP1 expression was highest during the fourth flowering stage.AGL19 was also expressed in flowers,stems and leaves of two varieties.Lm-XL-AGL19 expression was highest during the sixth flowering stage,while Lm-AGL19 expression was highest during the fifth flowering stage.The expression levels of AGL19 in flowers were higher than that in buds.Conclusions: 1.The total RNA extracted could be used for transcriptome sequencing and could be used to clone the full length c DNA of AP1 and AGL19 genes in L.macranthoides.According to bioinformatics predictions,they both belonged to MADS-box family genes.2.The results of quantitative fluorescence PCR showed that the relative expression levels of AP1 and AGL19 genes in different flowering stages and organs of L.macranthoides were different.AP1 and AGL19 may be related to the special characteristics of Xianglei varieties,such as long bud period and non-expansion of corollas.3.These studies laid a foundation for further verifying the biological functions of AP1 and AGL19 genes,and provided a basis for exploring the internal causes of the special phenotype of Xianglei varieties.