A Preliminary Study On The Function Research Of Cell Wall Invertase Gene And Its Promoter Of Lilium Brownii Var.giganteum

As an important flower bulb,lily has a long cultivation history.It has a wide range of applications in edible,medicinal and ornamental aspects,and it is also known as the "king of bulbous flowers".With the continuous yearning for a better life in modern society,the demand for fresh cut flowers of lily is increasing year by year.At present,lily has become the third largest fresh cut flower in China after rose and chrysanthemum.However,the problem of rapid propagation of lily bulbs has not yet been effectively solved,which has a great impact on large-scale industrial production and commercial application of lily bulbs.Sucrose,as an essential nutrient for plant growth and development,also plays a crucial role in the initiation and development of lily bulbs.Lilium brownii var.giganteum was chosen as our experimental material,which is the variety of Lilium brownii.Based on our previous hybrid sequencing of Pac Bio and Illumina of L.brownii var.giganteum,through bioinformatics analysis,morphological observation and molecular experimental methods to further screen and study the cell wall invertase(CWIN)gene,which is the key gene during starch-sucrose metabolism.The full-length gene and its upstream promoter were cloned and preliminary functional verification.Therefore,our results could provide the theoretical background for clarify the correlation between bulblet proliferation and starch-sucrose metabolism.The main results are as follows:(1)Cloning and sequence analysis of cell wall invertase gene(LbgCWIN1)in L.brownii var.giganteumBased on transcriptome data,co-expression analysis and phylogenetic tree analysis combined with CWIN genes of several higher plants,the results showed that LbgCWIN gene was directly related to the expression pattern of stress resistance gene RPP13 and PATL5 gene,which is related to cell membrane material transport.The c DNA sequence of LbgCWIN was screened and found it was significantly differentially expressed in the bulbs of Lilium brownii var.giganteum treated with different concentrations of sucrose.By designing Primers,a 2928 bp genomic DNA sequence was cloned,which was named LbgCWIN1(Accession number MN794186),By using Prot Param,TMHMM,Swiss-Model software and other predictive analysis,574 amino acids were deduced and the specific 2-D and 3-D structure were obtained.The experimental result confirmed its cell wall subcellular localization.(2)Cloning and functional verification of the promoter of LbgCWIN1 geneChromosome walking method was used to clone the promoter of LbgCWIN1 gene,and a total of 2718 bp DNA sequences were obtained by two clones.Through the online promoter prediction software such as PLACE and Plant Care,multiple response elements were found in the promoter sequence,including abscisic acid(ABA)response elements ABRE and ABRE4,optical response elements ACE,AE-box,ATC-motif,Box 4,ERE and stress response elements TC-rich repeats,MYB-binding site and so on.Four missing promoter vectors,CWIN1-p1(2527 bp),CWIN1-p2(1606 bp),CWIN1-p3(904 bp)and CWIN1-p4(459 bp),were obtained by using the 5’-terminal deletion vectors.GUS staining and GUS activity of different 5 ’-terminated promoter vectors in tobacco leaves were obtained by Agrobacterium injection.It was found that the core structural region of the promoter of LbgCWIN1 gene existed within-459 bp,and there might be some enhancer and silencer sequences in its upstream region.(3)Construction and heterologous transformation of LbgCWIN1 gene overexpression vectorThe concentration of antibiotics for hygromycin resistance screening in Arabidopsis thaliana was 40 mg/L by observing its seed germination.Use seamless cloning methods to obtain the recombinant plasmid of the CDS of LbgCWIN1 gene and expression vector p CAMBIA 1301.The plasmid was transformed into wild-type Arabidopsis thaliana by the Agrobacterium-mediated floral dip method,and the hygromycin resistance screening,PCR positive screening and q RT-PCR method were used to confirm stable transgenic lines.The eventual homozygous T3 generation transgenic Arabidopsis were ontained.We,found that the excessive expression of LbgCWIN1 in Arabidopsis could partically affect the pod length of Arabidopsis which will enhance seed yield.