Light And Temperature Induction Of Trigger Bolting In Cabbage And Related Gene Cloning And Differential Expression Analysis

Bolting is an important and popular genetic character in Brassicaceae such as Brassica oleracea L.var.capitata L.First,in order to main the quality and production of cabbage in fields,it is necessary to adjust the sowing period or select varieties with appropriate mature feature to avoid early bolting caused by unsuitable sowing period or environment stimulus.Second,in order to add propagation genetations,shorten breeding period and speed up breeding process,it is nessary to promote bolting and flowering.Thirdly,In order to make parents flowering at the same time,it is necessary to mediate and control conformance of bolting and flowering of parent plants.Therefore, bolting was the key period and inevitable stage from vegetative development to reproductive development.Researching mechanism of bolting is of great importance for cultivation,breeding and hybridization seed production.Like flowering,if bolting,a complex reproduction development character,obviously appear,it is diffcult to control the tendency.Howerer,researching the mechanism of startup bolting(bolting potential,but not bolting) probably provides effective strategy to control bolting in essence.Both startup bolting and flower bud differentiation were inevitable stages of flowering transition in Brassicaceae and the flowering process of Brassicaceae plants were divided two stages. In stageâ… ,shoot aprical meristem transformed from vegetative growth(vegetative meristem,VM) to reproduction growth(inflorenscence meristem,IM).In stageâ…¡,IM transformed to floral meristem(FM) differentiated a full flower later.Startup bolting was the transformation from VM to IM,while flower bud differentiation was the transformation from IM to FM in cabbage.But it not was reported how the special charater of bolting triggered in cabbage.Repeated reliable and effective bolting-inducing systems in cabbage were found by quadratic orthogonal rotation combination design firstly,which provided a platform to further research the mechanism of trigger bolting,mediation of flowering time,development of pistil and stamen, interaction between stigma and pollen,mechanism of male sterile and self-incompatibility.Then the mechanism of biochemical response of bolting and related gene differential expression was researched and revealed,which provided foundation for life essence and cell fate from VM to IM in cabbage.At last,LFY_ZQ was cloned and analyzed,and it was related to vernalization type,which provided another strategy for classification of vernalization type,identification of bolting type and its mechanism research.The results were as follows.1.Bolting-inducing systems for cabbage cultivar ZQ with temperature and photoperiodThe three factor(temperature,photoperiod and treatment time) quadratic orthogonal rotation combination design was adopted to screen and establish the bolting-inducing systems for cabbage cultivar ZQ.It was treated in RXZ-300D intelligent controlling climate chamber that plants bottom stems inserted first euphylis were 5.5-5.6mm in diameter.The regressive equation of bolting in cabbage was y_ZQ=4.266×10^-2+1.755×10^-3x₂x₃-4.73×10^-3x₁x₂.The optimum bolting systems were obtained after optimization between stable temperature treatments(4℃/16h light and 4℃/8h darkness) and alternation temperature treatments(7℃/16h light and 4℃/8h darkness).Bolting was rapidly induced at a rate of 100%in 6 days in re-culture(20℃/16h light and 18℃/8h darkness) in cabbage pre-treated for 65 days under the conditions of 16-hour-long light and 4℃/7℃alternation temperature.Obvious increasing distance of adjacent leaves in plants center pole was found at that time,as well as elongating cabbage bolting and green little dot bumps in stem apex.Four days later, cabbage bolting reached the vegetables mouth sites and green little dot bumps grew to buds.Such two propagating patterns of Brassica oleracea L.var.capitata L.as several generations of one year and several generations of one plant were researched in the bolting-inducing system with temperature and photoperiod.Two generations could complete in a year with seed propagating pattern and each one was lasted for approximate 150d.At least three generations could succeed to finish in the propagating pattern of one plant which had the ability of bolting and flowering again after harvesting seeds inflorescences were cut.2.Biochemical mechanism of startup bolting of cabbageIn order to study the physiological and biochemical mechanisms of startup bolting of cabbage variety "ZQ",during the initial stage of bolting(re-cultivation 0-6 days) eight indexes(soluble protein,soluble sugar,free amino acid,starch synthesis enzyme,Vc,POD,SOD and CAT)were measured.Factor analysis with spss13.0 showed two substance-metabolizing leap-periods,i.e.2-3 days and 4-5 days after alternation low temperature treatment,in cabbage.Correlation analysis showed a super-relateness(α≤0.01) between soluble protein and Vc(negative correlation coefficient was -0.866),between POD and soluble sugar(positive correlation coefficient was 0.862), between POD and soluble amino(negative correlation coefficient was -0.899) and between starch synthesis enzyme and CAT(positive correlation coefficient was 0.973),and a strong relateness (0.01＜α≤0.05) between soluble protein and free amino acid(positive correlation coefficient was 0.755) and between starch synthesis enzyme and free amoni acid(positive correlation coefficient was 0.674),and a weak relateness(0.05＜α≤0.1) between free amino acid and soluble sugar (negative correlation coefficient was -0.566) and between POD and soluble protein(negative correlation coefficient was -0.631).Principal component analysis divided the eight indexes into three groups of principal components.The first group involved five indexes:soluble protein, soluble sugar,free amino acid,POD and Vc,which were probably in close relation to startup bolting of cabbage.Two indexes(CAT and starch synthesis enzyme) in the second group were probably in close relation to adversity resistance.One index(SOD) in the third group was probably in close relation to senescence resistance.During initial stage of bolting in cabbage,soluble protein content and free amino acid were gradually increased,while soluble sugar and POD were first decreased then increased.Vc content was first decreased then increased lastly decreased.3.Differential expression of trigger-bolting-related genes in cabbage stem apexCabbage cultivar "ZQ" was taken as materials to optimize cDNA-AFLP conditions including concentrations of template,Taq enzyme,Mg²⁺ and dNTPs.And after random testing selection amplified production through electrophoresis in 1%agarose and 6%polyacrylamide,at the same time,after reclaiming and validating different expression cDNA fragments,stable and credible system of cDNA-AFLP was established.Excellent results could be gained in 25μL PCR reaction supplemented with 40×-diluted pre-amplification template,1.2mmol/L Mg²⁺,0.2 mmol/L dNTPs, 0.04U/μL Taq enzyme.In order to research trigger-bolting-related genes of cabbage,cDNA-AFLP approach was employed to identify genes differentially expressed in cabbage stem apex after and before trigger bolting.Sixty-four primer combinations were used for selective amplification and seventy-six TDFs were selected for their differential expression under trigger bolting.Among the 76 TDFs,67 TDFs were up-regulated and 9 TDFs were suppressed.Fifteen TDFs were cloned and sequenced.Compared with the publicly available databases,15 TDFs were grouped into four bolting-related systems according to gene homology,system of hormone cascade reaction (T5A6-2,T5A5-2),system of oxidoreductases(T7A2-2,T8A6-1,T2A7-2),system of chloroplast and mitochondria(T8A2-3,T5A8-1,T2A7-3,T6A6,T8A6-2,T8A3-3),and system of mineral nutrition response reaction(T8A3-2,T2A7-1,T5A3,T5A4). 4.Cloning analysis and expression of LFY_ZQ in startup boltingDNA and RNA were extracted from stem apex of startup bolting in cabbage cultivar "ZQ" and LFY_ZQ gene was cloned by RT-PCR respectively with primer pairs 1 and 3.The LFY_ZQ fragment Cloned by primer pairs 1 from DNA and RNA respectively were 1348kb and 834kb.While LFY_ZQ fragment cloned by primer pairs 3 from DNA and RNA respectively were 1317bp and 510bp. LFY_ZQ gene of DNA and cDNA sequence fragments assembly were respectively 2560bp and 1239bp.Analysis of the nucleotide sequence by BLAST on line indicated that homology of LFY_ZQ was up to 91%between Brassica oleracea L.var.capitata L.and Brassica oleracea L.var.botrytis L.,up to 87%between it and Arabidopsis thaliana,up to 86%between it and Brassica juncea Coss., and up to 87%between it and Brassica Raphanus sativus L..Analysis of gene structure by DNAstar indicated that LFY_ZQ included three extrons(452bp,394bp,393bp) which totally coded 412 amino acids and two introns(514bp,807bp) which were unanimous with nucleotide splicing rules of GT-AG.And comparing amino acids coded by LFY_ZQ three extrons in cabbage with B.oleracea var. botrytims L.,B.juncea Coss.,Arabidopsis thaliana,B.Raphanus sativus L.showed that the conserved quantity of amino acids coded by extron 3 was higher than that of extron 1 and that of extron1 was higher than that of extron 2,and the conserved quantity of two sides amino acids of intron 2 was higher than that of intron 1.Analysis of amino acids among LFY_ZQ and other 12 kinds of Brassicaceae LFY on line were divided into two classes:classâ… with three green body vernalization plants(Brassica oleracea var. botrytis L.,Brassica oleracea var.capitata L.,Jonopsidium acaule) and classâ…¡with ten kinds of seed vernalization plants(Arabidopsis thaliana.,Brassica juncea Coss.,Barbarea vulgaris,Selenia aurea voucher E.Lyons,Boechera drummondii voucher R.Price,Capsella bursa-pastoris,Stanleya pinnata,Streptanthus glandulosus,Idahoa scapigera,Brassica Raphanus sativus L.).Phylogenetic analysis of LFY speculated that LFY was related to types of plant vernalization.Molecular relativity quality of LFY_ZQ was 46kD,the theoretical PI was 6.87,and atomic composition was C₂₀₁₉H₃₁₅₇N₅₉₉O₆₀₅S₁₆.LFY_ZQ was an unstable protein with stability coefficient 50.89.LFY_ZQ also was a hydrophobic protein with aliphatic index 72.77 and grand average of hydropathicity(GRAVY) -0.565.LFY_ZQ shared four kinds of active sites:N-myristoylation site, Protein kinase C phosphorylation site,Casein kinaseâ…¡phosphorylation site and Amidation site. N-myristoylation site focused on N side of LFY_ZQ and the other three kinds of activity sites mainly distributed the middle and C side of LFY_ZQ.Semi-quantitative RT-PCR indicated that the expression of LFY_ZQ was weak in 0-2d of startup bolting and became gradually stronger from 3d to 6d than that of former.