大豆半矮秆基因精细定位、基因克隆和功能分析

The plant height of soybean is an important trait associated with soybean yield. The reports in cereal crops have indicated that semi-height of plant was available to increase the seed yields under dense planting cultivation. They spawned the so-called Green Revolution in food production. Similarly, semi-dwarf soybean could gain more yield with the dense planting cultivation. For example, it was reported that soybean yield reached 361 kg/acre in Ohio in 1999. While the yield of soybean was only 110kg/acre to 130kg/acre in most of china. Soybean yield could be increased by planting semi-dwarf soybean varieties accompanied with the dense planting cultivation. However, the plant height of soybean links to some undesirable traits, which causes low yield and the linkage is difficulty broken by hybridization with other cultivars. Now, marker-assistant selection and gene manipulation will be the effective ways to overcome this barrier. Recombinant inbred lines (RILs) derived from the cross of L11 (semi-height) × L594 (tall) were used for bulked segregant analysis (BSA), consisted of high and low bulks (30 RILs each) for plant height. SLAF-tags were sequenced between the two parents and the two bulks to discovery polymorphisms and calculate allele frequency differences through Specific Length Amplified Fragment Sequencing (SLAF-seq) technology. New SNP and SSR markers identified with nucleic acid sequencing method accompanied with other markers from Soybase website were used to detect dwarf gene in F2 population and BC2F2 population. A 70 kb chromosomal region located on L was detected between Sat_099-Sat_113 comprising of ethylene related transcription factor and transcription related GA synthesis. To identify dwarf genes in Charleston, a list of genes within the hot regions of the genome generated through association analysis was scanned by gene prediction and gene annotation. Simultaneously, RNA of young leaves of V1, V10 and R2 developmental stages were extracted and sequenced using Illumina/Solexa Genome Analyzer. Only the genes related to plant height were considered as credible candidate genes. For a further determination of the candidate genes, QRT-PCR analysis was conducted using cDNA generated from total RNA of dwarf and normal lines including Dongnong 594, Charleston and other selected dwarf and normal materials. The genes upgradly expressed were cloned and transferred into Arabidopsis and soybean for functional analyses. cDNA Library from Charleston DNA was screened using yeast two-hybrid system to identify protein interacted with semi-dwarf gene. Then, binding assay in vitro was conducted to verify the interaction between semi-dwarf gene and the protein screened. Subsequently, the genes will transfer into soybean to generate semi-height germplasm with higher yield potential.

半矮秆大豆结合密植栽培技术能够获得高产。克隆半矮秆基因并明确调控机制，结合分子标记和转基因技术打破与不利基因连锁，能够提高育种效率和拓宽遗传基础。前期利用SLAF-seq技术开发的SNP标记结合公共图谱的SSR和InDel标记，将半矮秆基因定位于L连锁群200kb的基因组区域。本研究计划构建目标区段剩余杂合系，精细定位该基因，并克隆大豆半矮秆基因，再利用Gateway技术转化拟南芥和大豆，研究候选基因功能；同时，利用RNA-seq技术测定亲本及高矮秆池的不同发育阶段的生长点基因和种类差异，结合内外源激素施用测定及相应表型变化，对该基因功能和网络进行深入验证和分析；再利用酵母双杂交技术及双分子荧光互补分析技术分析互作蛋白，结合Aquaria蛋白调控位点可视化软件分析其功能；同时，开发该基因的功能标记，并通过标记辅助选择及转基因技术创制综合性状优良的半矮秆大豆新种质，进行分子辅助育种尝试。

半矮秆大豆结合密植栽培技术能够获得高产。但该性状基因与不利性状基因连锁，传统育种难以选育优良株型的大豆品种。基因克隆技术能够分析基因功能，结合分子辅助育种技术能够有效提高优质高产大豆新品种培育效率。本研究利用二代测序技术测定亲本及RIL和F2后代的高矮秆池DNA序列，构建了包括InDel、SNP等标记的高密度遗传图谱，并对控制株高的基因进行定位。QTL区间SSR1163-SSR1165在大豆物理图谱上只有一个大豆生长发育重要调控基因AUX28，是大豆生长素负调控转录因子。该基因转化拟南芥后代株高显著低于对照，同时大豆内外源激素含量分析结果也证明该基因是大豆主茎发育的关键基因，同时该基因明显提高拟南芥转化苗的耐盐性和耐旱性；利用生物大分子相互作用系统分析和该序列特异结合的转录因子，再利用LC-MS/MS质谱鉴定转录因子结果表明AUX28通过泛素化调控大豆主茎发育，是生长素信号途径的关键调控方式。同时本研究还克隆了在大豆营养生长旺盛时期在亲本及高矮秆池间差异表达的XTH91基因并转化拟南芥和大豆进行功能分析，过表达XTH91基因大豆株高显著高于对照，同时该基因和大豆苗期耐逆性密切相关。.项目组将102份黑龙江省不同育种时期的骨干亲本和国外种质资源导入到ms1和ms6不育系，通过自然授粉和蜜蜂辅助授粉方式提高大豆异交率以创制大豆新的优异种质资源。利用6个特异的InDel和SSR标记对不育系后代进行产量及表型的分子标记筛选，得到和亲本差异显著的高产株系，表明这些特异标记能够为大豆产量提高提供理论和材料支持。.通过项目实施，构建了1张高精细定位图谱，开发与大豆株高相关的主效QTL位点4-5个；明确生长素梯度调控大豆茎生长，具体调控机制是下一步研究重点。克隆AUX28和GmXTH91两个大豆株高调控基因；获得15份转半矮秆基因大豆新种质；通过分子辅助选择，获得半矮秆，综合性状优良的新材料11份；审定大豆新品种2个；发表SCI收录论文1篇，国内核心期刊发表论文2篇；通过本课题实施，培养访问学者2名，博士研究生1名，硕士研究生5名本科生10人；申请发明专利1项，获得黑龙江省科技进步二等奖1项。