水稻穗部性状QTL定位及候选基因分析

水稻(Oryza sativa)穗部性状与产量直接相关,其相关基因的挖掘与功能解析对于保障国家粮食安全意义重大。以籼稻华占(HZ)和粳稻热研2号(Nekken2)及构建的120个重组自交系(RILs)为实验材料,测定了穗长、每穗粒数、结实率、柱头外露率及一次枝梗数等穗部性状。结合高密度分子遗传图谱进行QTL定位,结果共检测到31个QTLs,分别位于第1、2、3、4、5、6、10和11号染色体上,其中2个位点的LOD值分别高达5.45与5.28。通过分析筛选QTL区间内可能影响穗部性状的相关基因,并利用qRT-PCR进行基因表达检测,发现LOC＿Os05g05490、LOC＿Os05g06150、LOC＿Os03g11700、LOC＿Os03g12430、LOC＿Os05g28720、LOC＿Os05g30890、LOC＿Os05g31740和LOC＿Os02g17880在双亲间的表达水平差异显著。其中,前5个基因编码三角状五肽重复蛋白,而后3个基因编码糖基转移酶。研究挖掘到31个与穗部性状相关的QTLs,为进一步定位和克隆相关基因,从而选育高产水稻新品种奠定理论基础。     

酵母脂肪酶分子进化及催化合成(S)-1,4-苯并二口恶烷*

Objective: 1,4-Benzodioxane is an important chiral intermediate for antihypertensive (Proroxan and Doxazosin), antidepressant (MCK-242) and other drugs, and it displays a broad spectrum of applications in the pharmaceutical field. Currently, in spite of high-yield advantage of chemical synthesis, there are some problems of environmental pollution and low production safety. Using lipase to catalyze synthesis of 1,4-benzodioxane provides a new pathway of green synthesis of 1,4-benzodioxane. However, natural enzymes face the dilemma of poor enantioselectivity. Therefore, molecular evolution was performed on Candida antarctica lipase B, and a technical route for the catalytic synthesis of 1,4-benzodioxane was established. Methods: Firstly, the key amino acid residues involved in substrate binding and conversion in the active center of Candida antarctica lipase B were analyzed, and saturation mutagenesis libraries on the interaction sites were constructed. Improved mutants with high efficiency and high enantioselectivity were then obtained using HPLC detection. Furthermore, catalytic synthesis conditions of mutant D223N/A225K were systematically optimized. Results: The results indicated that the mutants mainly derived from the pairwise site D223/A225 (such as D223N/A225K and D223G/A225W) were biased towards the synthesis of (S)-isoforms, while most of the mutants derived from the pairwise site E188/I189 (such as E188D/I189M) showed a bias for the synthesis of (R)-isoforms. Compared with WT, the ee_s value of the best mutant D223N/A225K to synthesize (S)-1,4-benzodioxane was increased from 11.9% to 29.3%. After systematic optimization of the reaction conditions, an ee_s value of (93.9±0.16)% and a conversion rate of (47.5±2.33)% were achieved using mutant D223N/A225K to catalyze kinetic resolution of methyl (R,S)-2,3-dihydro-1,4-benzodioxin-2-carboxylate in n-butanol/phosphate buffered saline (20∶80, V/V) biphasic solvent at 37℃ for 50 min. Conclusion: An efficient kinetic resolution of methyl (R,S)-2,3-dihydro-1,4-benzodioxin-2-carboxylate was successfully achieved by molecular evolution and optimization of conditions, which provides a new example for the creation of new enzymes by protein engineering technology, and also provides a theoretical and technical foundation for the efficient synthesis of (S)-1,4-benzodioxane molecules by enzymatic methods.