| Plant mutants are valuable materials in genetics research.As the birch(Betula platyphylla Suk.)genome sequencing has been completed,it is possible to study on the T-DNA insertion birch mutants and subsequently identify mutated genes.In this study,the mutant phenotypes of a T-DNA birch mutant br were identified,and the flanking sequences of T-DNA insertion sites were isolated to identify the mutated gene.Meanwhile,the function research of mutated gene was carried out.The research results are as follows:(1)The wild type WT,BpCCR1 overexpression transgenic birch line OE2 were used as control to analyze the changes of growth traits,plant architecture,photosynthetic capacity,plant disease susceptibility,and endogenous plant hormones content and other characters in br.The results showed that the tree height and ground diameter of br were significantly less than those of two control lines.The plant architecture of br exhibited weaker and multiple lateral branches,small internode length and small branches angle.The relative chlorophyll content,photosynthetic parameters and chlorophyll fluorescence parameters analysis showed that the relative chlorophyll content and non-photochemical quenching coefficient of br were low,while the stomatal conductance and transpiration rate were high.The disease susceptibility analysis showed that br was more susceptible to Alternaria alternata.The results of endogenous plant hormones contents showed that auxin and Zeatin content in br main and lateral branches' shoots were similar,while the ratio of zeatin content to auxin content was higher in br than those of WT and OE2.In addition,the jasmonic acid content was low and the salicylic acid content was high in br compared to WT and OE2.(2)Transcriptome analysis of WT,OE2,and br main and lateral branches' apical shoots showed that 406 genes(149 up-regulated and 257 down-regulated)were found to be differentially expressed in br main branches' apical shoots,and 396 genes(125 up-regulated and 271 down-regulated)were found to be differentially expressed in br lateral branches'apical shoots.These differentially expressed genes(DEGs)were mainly involved in flower development,plant fertility,plant growth and development,organ morphogenesis,shoot apical meristems activity,plant hormones biosynthesis and signal transduction.(3)Two T-DNA insertion sites were identified in br genome by TAIL-PCR,second-and third-generation whole genome re-sequencing techniques.One of two sites was located on Chr 5(1729009?1729377 bp),resulted in 369 bp heterozygous deletion containing 122 bp 5'UTR and 247 bp first exon of BpCOI1 gene(Bpev01.c0817.g0010.m0001).The results of qRT-PCR showed that the expression of BpCOI1 in br was significantly lower than that in WT and OE2,and the ability of br to respond to exogenous MeJA was decreased.(4)The BpCOI1 promoter was transformed into birch via Agrobacterium-mediated method,and four ProBpCOI1::GUS transgenic lines were obtained.The analysis of GUS staining and GUS gene expression showed that the BpCOI1 transcriptional activity could be detected in roots,stems,leaves and buds,and GUS gene was highly expressed in stems and buds.Based on predicted cis-acting elements,the ProBpCOI1::GUS transgenic line was subjected to IAA,ABA,GA,SA,MeJA,ET and photoperiod treatments.The results of GUS activity and GUS gene expression showed that BpCOI1 promoter had different responses to all these plant hormones,while it was significantly induced by MeJA.The BpCOI1 promoter activity was also affected by different photoperiod treatments,and BpCOI1 promoter activity could be promoted by long light and inhibited by short light.(5)The BpCOI1 was located on nucleus,and a synonymous mutation in 150th base was detected in birch compared to reference genome.The multiple sequence alignment of proteins from birch,Arabidopsis,rice and poplar showed that the 1-40 amino acids contained part of F-box domain of COI1 in different species were not conservative.The results of yeast two hybrid and bimolecular fluorescence complementary showed that this nonconservative sequence of F-box did not affect the protein interactions between BpCOI1 and SKP1 family.(6)The Agrobacterium-mediated transformation procedure was retrieved for the generation of BpCOI1 transgenic birch lines,and eight 35S::BpCOI1 overexpression transgenic birch lines were obtained.Two fragments were interfered by RNAi,one fragment was located on the third exon of BpCOI1,and the other fragment was located on the first exon of BpCOI1 containing the part of br deletion region.Five 35S-BpCOI1-RNAi BpCOI1 suppression transgenic birch lines were obtained,respectively.Compared to wild type,the growth traits such as tree height,ground diameter,branches number of one-and two-year-old BpCOI1 transgenic lines did not change regularly.The MeJA treatments analysis showed that high concentration of MeJA inhibited the roots growth of wild type birch lines and BpCOI1 overexpression transgenic lines,while it did not significantly inhibit the roots growth of BpCOI1 suppression transgenic lines.The susceptibility analysis showed that the disease incidence rate of BpCOI1 suppression transgenic birch lines were more than 83.33%,and the range of leaf disease index were from 10.51%to 22.95%after treating with A.alternata spore suspension for 7 d,while no disease spots were found in the leaves of BpCOI1 overexpression transgenic and wild type birch lines.The qRT-PCR results showed that BpCOI1 and BpMYC2 were up-regulated in BpCOI1 overexpression transgenic birch line compared to wild type after infection by A.alternata,while these two genes were not up-regulated or up-regulated insignificantly in BpCOI1 suppression transgenic birch lines.(7)Transcriptome analysis of BpCOI1 transgenic lines and wild type showed that 1385 up-regulated DEGs and 1137 down-regulated DEGs were found in BpCOI1 overexpression transgenic birch line,while 1446 up-regulated DEGs and 921 down-regulated DEGs were found in BpCOI1 suppression transgenic birch line compared to wild type.Most of genes involved in terpenoids and secondary metabolite biosynthesis and four genes involved in JA signal transduction were down-regulated in BpCOI1 suppression transgenic line,while six genes involved in SA transduction signal,most of genes involved in plant-pathogen interaction and plant senescence regulation,and genes belong to WRKY transcription factor family were up-regulated.Besides,genes involved in other plant hormones biosynthesis and transduction signal,a large number of plant hormones-,stress-,fungus-,and bacteria-responsive genes were also differentially expressed in BpCOI1 overexpression and suppression transgenic lines.In summary,in this study,a T-DNA insertion mutant br with multiple phenotypic changes such as susceptibility to disease,multiple-branches,dwarf was characterized,and T-DNA insertion sites in mutant were identified.Then,the mutant gene BpCOI1 was isolated to carry out function analysis of BpCOI1 gene and promoter.These results showed that COI1 protein was essential to JAs perception,a reduced expression of BpCOI1 could lead birch to be insensitive to MeJA,and more susceptible to plant disease.These results may provide references for resistance breeding of birch. |
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