陆地棉巯基蛋白酶抑制剂基因克隆及其氨基酸序列分析

通过对植物中已知巯基蛋白酶抑制剂(cystatin)的保守性分析,设计1对简并引物,从陆地棉栽培种中棉所29(GossypiumhirsutumL.cv.Zhongmiansuo29)cDNA中克隆出1条巯基蛋白酶抑制剂基因片断,经测序和对测序结果在有关数据库中检索分析,发现该片段与1条中棉(G.arboreumL.)EST及1条雷蒙德氏棉(G.raimondiiL.)cDNA同源性高达96%。三者编码蛋白的氨基酸同源性达100%,且完全符合CPI的特征;所克隆基因片段的氨基酸序列与NBCI蛋白质数据库中登录的豇豆、向日葵、玉米、水稻中CPI均有高度同源性,表明该片段包含编码陆地棉CPI的完整序列。     

Response of cotton fiber quality to the carbohydrates in the leaf subtending the cotton boll

This study investigated the effect of nitrogen (N) fertilization on leaf and boll N and carbohydrate concentrations in the development of fiber quality. A two‐year field study was conducted with two cotton (Gossypium hirsutum L.) cultivars, Kemian 1 (average fiber strength 35 cN tex^–1) and NuCOTN 33B (average fiber strength 32 cN tex^–1) at five (2008) and four (2009) N levels. The relationship between leaf and boll N and carbohydrate concentrations was assessed from measurements of N, carbohydrates, chlorophyll (based on SPAD readings), and free amino acids in the leaf subtending the boll, together with fiber carbohydrates and development of fiber quality. Results indicate that leaf N concentration more accurately reflected boll N status than the concentration of chlorophyll or free amino acids. Leaf sucrose and nonstructural carbohydrate had a quadratic relation with leaf N concentration (p < 1%). The optimal leaf N concentration ranged from 3.0% to 2.4%. During 24–38 d post‐anthesis (DPA), fiber sucrose was positively related to leaf sucrose and nonstructural carbohydrate (p < 5%), but was not correlated with leaf starch or total soluble carbohydrates. Fiber strength was positively correlated with fiber sucrose before 38 DPA, and it appeared to be more easily influenced by the fiber sucrose concentration than fiber length, fineness, or maturity. These results suggest that 24–38 DPA is a crucial period for fiber development which might be significantly influenced by physiological and ecological factors. In addition, sucrose or nonstructural carbohydrates in the subtending leaf could be used as a monitoring index to evaluate sucrose levels in the developing fiber, and also for predicting the final fiber strength.     

陆地棉干旱胁迫响应基因GhGR的克隆及特征分析

【目的】克隆陆地棉干旱胁迫谷胱甘肽还原酶基因（GhGR）,并对其序列进行生物信息学分析和表达分析。【方法】利用RACE和RT-PCR技术克隆陆地棉谷胱甘肽还原酶基因的全长序列,应用生物信息学软件对获得的基因序列及编码的蛋白序列进行分析;通过基因枪转化和实时荧光定量PCR表达对该基因表达部位和表达模式进行分析。【结果】从陆地棉（Gossypium hirsutum L.）中克隆了谷胱甘肽还原酶基因GhGR,cDNA全长1 035 bp,其中,ORF为792 bp,编码263个氨基酸。氨基酸序列比对和同源性分析显示该基因与杨树（XP_002299276.1）、蓖麻（XP_002518118.1）、葡萄（CAN74593.1）同源性最高,分别为90%、91%和91%。系统发育树结果显示,GhGR与葡萄中该蛋白的亲缘关系最近。基因枪转化和实时荧光定量PCR分析表明GhGR定位于洋葱的细胞膜和细胞核膜,并且其表达量受干旱胁迫诱导上调表达。【结论】从陆地棉克隆得到谷胱甘肽还原酶基因GhGR,初步认为该基因对干旱胁迫有一定响应。     

不同土壤湿度对棉花苗期生长及生理机制的影响

不同土壤湿度(15%、35%、55%、75%、95%、115%)对棉花(Gossypium hirsutum L.)苗期生长和若干生理指标的影响。试验结果表明,棉苗正常生长土壤适宜含水量为55%~75%,过多过少均不利于棉苗健壮生长。棉花苗期受水分胁迫,植株可溶性蛋白质含量、脯氨酸(Pro)含量增加;水分胁迫处理初期,超氧化物歧化酶(SOD)、过氧化物酶(POD)活性增强,随着胁迫时间的延长,抗氧化酶活性不断降低;植株真叶数、株高、茎粗以及干重和鲜重明显减少,根系干重和鲜重也同样降低。     

棉花MADS-box蛋白基因（GhMADS-13）的克隆和表达分析

一组具有MADS-box结构域的转录因子在控制花器官的诱导与发育中起着重要作,以中棉所36（CCRI）均一化全长cDNA文库为基础,结合EST测序分析,分离获得一个棉花的MADS-box基因全长cDNA。该基因cDNA全长1079bp,包含一个732bp的完整的开放阅读框,编码234个氨基酸,具有典型的MADS-box结构域和完整的K区,命名为GhMADS-13（GenBank：FJ409870）。与拟南芥、葡萄等植物的AGL6类基因具有高度的同源性。实时定量RT-PCR分析表明,GhMADS-13在CCRI36的花器官发育中早期表达量逐步增加,后期有下降趋势。推测GhMADS-13可能在开花诱导和花器官发育过程中起着重要作用。     

不同开花期棉铃干物质积累规律研究

本文通过对１９８８、１９８９两年棉铃发育规律的研究得出：不同开花期的棉铃各组成部分子物质积累均呈“Ｓ”型曲线，可用Ｌｏｇｉｓｔｉｃ方程加以拟合，效果良好，相关系数都在０．９以上。棉子的干物质积累还可用直线方程模拟，效果更好，相关系数在０．９８以上。其中铃壳的干物质积累较快，最快时期在花后１０～１５ｄ，花后３０ｄ达最大值，但是大积累量、生长特征值随着开花期的向后推迟而逐渐增大；而棉子和棉纤维的干物质积累要延续到花后４５～５０ｄ才达到最大值，其增长速度最快的时期在花后２０～３０ｄ，它们的干物质最大积累量、生长特征值、旺盛生长期的长短是以中期开花的较高，前期和后期开花的较低。     

棉花海陆杂交纤维品质性状遗传参数分析

为了研究棉花海陆杂交纤维品质性状的遗传规律,本试验采用5个陆地棉品种（系）作母本与5个海岛棉品种（系）杂交,配制25个杂交组合,随机区组试验设计,重复2次。主要检测杂交种F1代纤维品质性状,以小区平均数进行相应的方差分析及遗传参数估计。结果表明：陆地棉亲本间变异除马克隆值外,其他纤维品质性状均差异显著或极显著,而海岛棉亲本间除伸长率外,其他性状均存在显著或极显著差异;各纤维品质性状双亲互作效应均不显著。另外,遗传参数结果表明：绒长、比强度和马克隆值的遗传变异系数与表型变异系数变化趋势基本吻合,且均大于环境变异系数。整齐度和伸长率的遗传变异系数均小于环境变异系数,说明整齐度和伸长率的变异中环境因素的作用不容忽视。绒长、比强度和马克隆值的广义遗传率较高,在采用海陆杂交改良棉花纤维品质性状时,适宜在早代选择优异株系。     

Somatic Embryogenesis and Plant Regeneration from Two Recalcitrant Genotypes of Gossypium hirsutum L.

An improved protocol has been developed for somatic embryogenesis and plant regeneration of recalcitrant cotton cultivars. High callus frequencies and embryogenic tissue were developed in MSB medium supplemented with gradient concentrations of KT and 2,4-D, their concentration decreasing from 0.1 to 0.01 mg L⁻¹. Somatic embryos were successfully incubated in 1/2 macronutrient MSB suspension supplemented with 0.5 g L⁻¹ glutamine and 0.5 g L⁻¹ asparagine. Decrease in macronutrient concentration of MSB significantly alleviated browning and was beneficial to suspension cells. Transformation of somatic embryos into plants was induced in MSB medium supplemented with 3% sucrose, 0.5 g L⁻¹ glutamine, 0.5 g L⁻¹ asparagine, and 6.0 g L⁻¹ agar. The effect of sucrose as carbohydrate was better than that of glucose for plant germination. Using this protocol, regenerated plantlets from the CCRI521 and Zhongzhi86-6 reached to as much as 19.6 and 18.5% somatic embryos, respectively.     

不同棉花品种棉纤维发育过程中生化物质的比较

选用陆地棉品种中棉42和新陆早36为材料,测定了开花后棉纤维发育过程中可溶性糖和可溶性蛋白质含量及相关酶活性的动态变化。结果表明,棉纤维发育过程中两品种的可溶性蛋白质含量在开花后7～21 d急剧下降,其中中棉42在开花21 d后趋于平缓,新陆早36在开花21 d后仍下降明显,在开花后28 d才趋于平缓;可溶性糖含量在开花后7～14 d下降,至开花后14 d又急剧上升,开花后21d时达到最大值,随后又开始快速下降,开花后35 d时可溶性糖含量最小,此后趋于平缓;过氧化物酶（POD）活性总体呈上升趋势;吲哚乙酸氧化酶（IAAO）活性在开花后7 d较低,开花后14 d和28 d左右是棉铃发育中 IAAO活性的峰值,开花35d后两品种棉花 IAAO活性都在降低并且变化趋于一致。     

陆地棉锌指蛋白(GZFP)的原核表达

采用大肠杆菌表达体系来获得陆地棉锌指蛋白（GZFP）的融合蛋白．以pMD18-GZFP质粒为模板,用PCR方法扩增获得GZFP基因的编码区序列,将其克隆到表达载体pET28b（＋）中,转化宿主菌BL21（DE3）,成功地构建了陆地棉GZFP基因原核表达载体pET—GZFP、经IPTG诱导、SDS-PAGE电泳分析和蛋白质杂交检测表明,陆地棉GZFP以融合蛋白的形式在大肠杆菌中得到大量表达．