甜瓜属野生种铝胁迫诱导基因ChAI的克隆及序列分析

应用同源序列克隆策略从铝盐胁迫处理的甜瓜属野生种(Cucumis hystrix Chakr.) 叶片中获得了612bp的片段,通过在EST数据库进行同源检索,发现一条甜瓜EST序列（GenBank登录号：AM724963.2）与之高度同源,据此设计引物并经RT-PCR扩增和序列拼接,最后获得了铝诱导基因cDNA序列全长,命名为ChAI (GenBank登录号：FJ968438 )。序列分析表明该基因全长967bp,其中开放读码框(ORF)长711bp编码236个氨基酸组成的多肽,5′ 和3′ 端非编码区长度各为15bp和241bp。DNAMAN同源性分析表明该基因与葡萄、棉花、羊乳（轮叶党参）、白骨壤（真红树植物）、拟南芥等植物的铝诱导蛋白的氨基酸序列具有78.6%以上的同源性。运用半定量RT-PCR技术对ChAI 基因的转录水平进行分析,根据该基因在铝胁迫下大量表达推测ChAI 与耐铝盐胁迫响应相关,但具体耐铝胁迫机制还有待深入研究。     

中国野生葡萄抗白粉病基因RAPD标记的克隆及序列分析

对获得的中国野生葡萄抗白粉病基因RAPD标记OPW02-1756、OPO11-964、OPY13- 661、OPB11- 520、OPW05-766、OPV03-1365和OPJ16-759进行了回收、克隆、测序及序列分析。结果表明,7个RAPD标记的实际长度分别是1756bp、964bp、661bp、520bp、766bp、1365bp和759bp。OPW02-1756序列与3条欧洲葡萄cDNA克隆获得的EST序列有94 ~ 98%的同源性。OPO11- 964序列与欧洲葡萄“赤霞珠”的1条EST序列有93%同源性,与“霞多丽”的2条果实不同发育时期获得的EST序列有90%和87%的同源性,与甜橙在多种病原菌侵染后通过cDNA文库获得的1条EST序列有83%的同源性。OPO11-964最大的一个阅读框架包含444个碱基,编码147个氨基酸,该氨基酸序列和21个其它生物的未知功能蛋白序列有部分相似性。OPY13-661序列与欧洲葡萄16条EST序列有同源性,其中与“赤霞珠”cDNA克隆的EST有较高的同源性, 与“霞多丽”的2条叶片非生物胁迫有关的EST序列同源性均为89%。OPB11-520序列与拟南芥基因组4条未知功能DNA序列有94%同源性。OPW05-766序列与来自于酿酒葡萄的GAG-POL 前提有50%的同源性。OPV03-1365序列与水稻基因组6条序列有81 ~ 88%的同源性,与拟南芥基因组3条序列有84 ~ 91%的同源性。OPJ16-759与欧洲葡萄“霞多丽”叶片14条非生物胁迫EST序列有83 ~ 100%的同源性,与欧洲葡萄“Cabsau”浆果3条水分胁迫的EST序列有94 ~ 100%的同源性。     

大梅与梅山猪背最长肌正反消减文库的构建及序列分析

为从基因差异表达的角度研究杂种优势的分子机理,构建了差减效率均为210倍的大梅与梅山猪背最长肌正反消减文库。在大梅(被消减)-梅山库中挑选了610个有效克隆,经斑点杂交筛选出48个阳性克隆,测序后经Blastn网上比对揭示了37个基因,其中28个EST是已报道基因的cDNA片段,9个在NCBI数据库中没有同源序列。在梅山(被消减)-大梅挑选了580个有效克隆,经斑点杂交筛选出45个阳性克隆,测序后经Blastn网上比对揭示了34个基因,其中26个EST是已报道基因的cDNA片段,8个在NCBI数据库中没有同源序列。     

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。     

辐照花粉辅助授粉克服百合受精前障碍的研究

采用60Co辐照LA杂种系百合‘耀眼’(‘Dazzle’)花粉,通过辅助授粉克服百合‘Dazzle’(♀)×川百合(Lilium davidii)的远缘杂交障碍,采用荧光显微技术观察授粉后3~7d父本花粉及花粉管在母本雌蕊中的行为。研究结果表明:采用未处理花粉直接授粉,花粉管在花柱中伸长到一定长度后形成胼胝质,停留在花柱中不能进入子房;采用蒙导花粉及先锋花粉授粉法能促进花粉管从花柱进入子房,并接近或可能进入胚珠,表明辐照花粉辅助授粉能部分地克服百合‘Dazzle’(♀)×川百合(Lilium davidii)远缘杂交的受精前障碍。     

结球白菜结球前期基因表达序列标签（EST）分析

以结球白菜(Brassica rapa L.ssp．pekinensis)结球前期心叶为材料，构建了结球前期球叶cDNA文库，随机挑选克隆单向单次测序后，共得到1162条峰图良好和插入片段长度大于150bp的可用序列。BLASTX及BLASTN序列比对分析表明，94．8％(1102／1162)的表达序列标签(EST)可在蛋白质或核苷酸水平上找到同源类似物。同源性最大的蛋白质按物种来源分析后发现，大约77％的功能已知蛋白质来自拟南芥(Arabidopsis thaliana)，另外还有60个EST与已发表的植物基因没有同源性，这一部分EST对于研究结球白菜独特的生理和形态发育途径以及开展基因组分子作图具有重要的意义。同源蛋白质按其功能进行分类表明，参与蛋白质合成过程的酶或蛋白质的EST数量最多，其次是参与能量代谢过程(包括光合作用)的EST序列。在核苷酸水平上，全部EST中只有51％的同源物来自拟南芥。对上述1162条EST进行片段重叠群分析(contig analysis)共获得895个非冗余片段重叠群，其中723个仅由一个EST组成(即singletons)，表明结球白菜结球过程中表达的基因种类繁多。大量EST的获得为进一步了解结球白菜结球机制及获取相关基因提供了重要的序列信息。     

玉米混合组织的EST文库的构建和部分EST序列的分析

以CLONTEECH的pDNR-LIB质粒为载体,构建了干旱和盐碱共胁迫48h和相应的无胁迫正常生长条件下耐旱玉米(ZeamaysL.)YQ7-96品系的雌雄分化期(12片叶龄)的混合组织(叶、茎和花器)EST文库。该文库容量>2×105个克隆,覆盖了预计的玉米基因组5万个基因的4倍以上。随机对3168个克隆中插入的cDNA进行测序,获得有效序列2098条,其中,2002个克隆中的cDNA进行了5'端测序,96个克隆中的cDNA的进行了5'和3'两端测序。结果表明,3'端测序的96个克隆的cDNA都含有polyA尾巴;进一步对这2098条组装分析,形成168个重叠群(Contig)和1693个单拷贝EST(Singlet),代表了共计1861条独立基因(Unigene);根据GenBank中的玉米EST数据库和BLASTn分析软件进行核苷酸同源性分析,1731条(93.01%)EST的score值大于或等于100,130条(6.99%)的EST的score值小于100,说明这130条属于新的表达基因的序列,有21条可以定位在玉米的不同染色体上;BLASTx分析表明,2098条EST中,1007条(48%)有功能注释信息,1091条(52%)无功能注释信息。该文库中的EST将会对公共数据库中现存的玉米EST数据起到补充作用。     

陕229干旱复水诱导基因表达及小麦乙烯受体(TaERS)基因特征分析

为了解小麦响应水分胁迫后复水条件下的基因表达特征,以小麦(Triticum aestivum L.)主栽品种陕229的3叶1心期幼苗为材料,采用消减杂交技术,构建了干旱复水条件下的SSH-cDNA表达文库。从消减文库中随机挑选59个插入片段大于400bp的阳性克隆测序,去除冗余序列和嵌合序列后,获得高质量EST序列32条(GenBank登录号为ES466767~ES466798)。序列比对分析表明,小麦干旱后复水的基因表达与植物对其他非生物胁迫的响应具有交叉性;17条EST序列与已知编码蛋白的基因同源性较高,涉及植物的信号传导、能量代谢、转录调控等方面;其他序列为新的EST。以其中一个与乙烯受体基因(ERS)同源性较高的EST序列为基础,采用同源克隆及RACE技术从小麦中分离了4个乙烯受体基因的全长cDNA序列,长度分别为2090、2271、2216和1886bp。4个全长cDNA序列所编码氨基酸序列的同源性极高(99%以上),且均具有ERS典型的GAF、HisKA和HATPase-c跨膜结构,分别命名为TaERS1(HM347272),TaERS2(HM601437),TaERS3(HM601438)和TaERS4(HQ111523)。植物ERS氨基酸序列多重比较表明,小麦与水稻的相似性最高(93%);TaERS基因的全长cDNA序列间比较共发现SNP位点23个。定量PCR表达分析显示,小麦TaERS家族基因参与了小麦植株响应水分胁迫和复水的调控机制。研究结果有助于进一步认识小麦干旱后复水的基因表达谱和小麦水分高效利用机制,探索小麦乙烯受体在水分高效利用中的作用。     

小麦品种小偃22干旱诱导基因及其表达分析

利用抑制消减杂交(SSH)技术构建了小麦(Triticum aestivum)品种小偃22干旱胁迫诱导基因表达的SSH-cDNA文库,从文库中随机挑选插入片段大于400 bp的阳性克隆60个进行测序,去除冗余序列和嵌合序列后,获得了31条高质量表达序列标签(EST)序列(GenBank登录号:ES466737～ES466766,ES466799).进一步采用RT-PCR对其中的胚胎形成晚期富集蛋白基因(LEA)和△1-吡咯啉-5-羧基合成酶基因(P5CS)的表达模式进行了分析.结果表明,获得的EST序列中的26条EST序列具有功能已知的同源基因,这些基因多数不仅与植物的非生物胁迫相关,也与植物的生物胁迫反应相关,表明植物在胁迫反应中有明显的交叉适应性,其中与小麦表达序列高度同源的EST占58%.24条EST序列与功能已知蛋白的同源性较高,主要涉及植物的信号传导、能量代谢、转录调控及防卫反应等.表达模式分析表明,LEA家族蛋白D9在土壤绝对含水量达到4.8%时上调表达,在萎蔫后复水2 h时表达量最高,随着复水时间延长表达量降低并趋于稳定;PSCS基因D16在渍水条件下、严重干旱胁迫下均上调表达,而在可维持生长的水分条件下和严重干旱后复水4 h后表达量急剧下降.序列分析表明,LEA蛋白基因D9与PSCS基因D16均是部分同源基因,暂命名为TaLEA-D9和TaP5CS-D16.     

钾高效基因型棉花的筛选及其生理机制的研究

通过营养液培养,设缺K和适K处理,进行棉花苗期培养,以苗期干物质的K效率系数(-K/ K)评价K效率差异,从86个不同系谱的棉花品种中分次逐步筛选,获得4个候选品种.对候选品种进行全生育期土培试验,设施K和不施K处理,获得皮棉产量,以皮棉产量的K效率系数(-K/ K)来反映品种间的K效率差异,确定103为K高效高潜力基因型,122为K低效低潜力基因型,163和165为K高效低潜力基因型.103具有较强的吸收土壤速效K、活化土壤缓效K的能力,并以较低的K含量往繁殖器官运输分配,建成较多的子棉和皮棉,这可能是其K高效的机制之一.     

外施吲哚乙酸对棉铃蔗糖代谢及产量性状的影响

为探究外施吲哚乙酸(IAA)对棉铃蔗糖代谢及棉铃产量性状的调控机制,以陆地棉(Gossypium hirsutum L.)品系A201为材料,用0.1 mg·L^-1 IAA溶液连续处理开花前1 d至开花后2 d的棉铃,以去离子水处理为对照,研究IAA处理对棉铃胚珠外表皮纤维细胞突起、蔗糖代谢及棉铃产量性状的影响。结果表明,IAA处理能够显著提高棉铃单铃重,促进棉铃开花当日纤维细胞启动的密度。与对照相比,IAA处理可增强开花后30 d种胚磷酸蔗糖合成酶(SPS)活性,导致蔗糖浓度升高,为种胚大分子物质合成提供更多的底物;并提高了开花后30~40 d种胚和开花后20~30 d种皮蔗糖合成酶(SS)的活性,增加了种胚和种皮的淀粉浓度,促进了棉花种子库物质合成。另外,IAA处理棉铃可以促进棉铃种胚和种皮蔗糖代谢关键酶的活性,提高其碳水化合物特别是淀粉的浓度,有利于棉铃种子的发育,这可能是导致单铃重提高的重要原因。本研究结果对于利用IAA提高棉花产量具有指导意义。     

不同量化RT-PCR的方法在棉花纤维GhCAD基因表达中的研究

以陆地棉（Gossypium hirsutum L.）高代品系CJ01在开花后5、10、15、20、25d（DPA）共5个不同发育时期的棉花纤维为材料,分别利用常规半定量RT-PCR法、QuantumRNA™18S内标相对定量法以及实时荧光定量 RT-PCR法,对棉花肉桂醇脱氢酶（GhCAD）基因在棉花纤维不同发育时期中的表达进行相对定量分析,并测定了内标表达的稳定性。结果表明3种方法获得相似的结果,GhCAD基因在5～15DPA时表达量较低,20、25 DPA时表达量升高。3种方法虽各有利弊,但均为研究基因量化表达的有力工具。     

Silicon and salicylic acid in the physiology and yield of cotton

Silicon (Si) and salicylic acid (SA) foliar applications can benefit cotton yield especially if there is stress during cultivation. The objective of this study was to evaluate the foliar application of Si and SA on the photosynthetic variables and cotton yield. The experimental design used was randomized complete block, constituted by Si foliar application in potassium and sodium balanced silicate form (0 and 3.6?g L^?1 of Si) and SA (0 and 210?mg L^?1). The treatments were applied in three leaf sprays during the reproductive stage that coincided with water stress in tillage. Therefore, the Si foliar application associated with SA favors the physiological variables, increasing the photosynthesis, stomatal conductance and water use efficiency reflecting on the increase of cotton yield.     

膜下滴灌条件下棉株温湿度微环境

通过对膜下滴灌棉花根区土壤温度、水分、冠层叶温、气温及冠层空气湿度的观测,分析了该灌水条件下棉花土壤水、热关系,冠层温、湿度关系,结果发现:棉花根区土壤水分与温度之比在深度方向上近似呈线性关系,冠层叶—气温差与空气饱和差也呈线性关系,说明膜下滴灌技术能够为棉花根系提供良好的吸水环境,有利于棉花生长。     

Long-Term Effects of Aluminum and Cadmium on Growth,Leaf Anatomy,and Photosynthetic Pigments of Cotton

Aluminum (Al) and cadmium (Cd) are two elements that contaminate soil in different ways as waste products of some industrial processes and that can be tolerated by some plant species in different concentrations. In this study, growth parameters of leaves and stems (fresh and dry weights, stem lengths, leaf surface area, and lamina thickness), anatomical changes in leaves (lower and upper epidermis, stomata and mesophyll tissue), and photosynthetic pigment contents (chlorophyll a and b, total chlorophyll, and carotenoids) were investigated in cotton (Gossypium hirsutum L. cv. Nazilli 84S), which was treated with Al and Cd for 3 months. Cotton seedlings were grown in greenhouse conditions and watered with Hoagland nutrient solutions, which contained 0, 100, and 200 μM aluminum chloride (AlCl₃) and cadmium chloride (CdCl₂). It was observed that reduced soil pH positively affected many parameters in cotton plants. Aluminum accumulation was greater in leaves than stems while the opposite was true for Cd accumulation. Leaves and stems of cotton plants treated with 100 and 200 μM Al and Cd showed slight growth changes; however, high concentrations of Al (200 μM) caused significant reductions in leaf area and leaf fresh weight, whereas stem fresh weight decreased with 200 μM Cd treatment. Anatomical parameters were mostly affected significantly under both concentrations of Al and Cd solutions (100 and 200 μM). The results revealed that the anatomical changes in the leaves varied in both treatments, and the long-term effect of the tested metals did not include harmful effects on anatomical structures. Moreover, the variations could be signals of tolerance or adaptive mechanisms of the leaves under the determined concentrations.     

The effect of starter fertilizers on cotton production in legume residues

Abstract

Field and greenhouse studies were conducted to identify starter fertilizers which would enhance cotton seedling survival, growth, and yield in legume residues. Field studies were initiated in the fall of 1982 on a Norfolk sandy loam (Typic Paleudult) in the Upper Coastal Plain of Alabama. Winter annual legumes, crimson clover (Trifolium incarnatum L.) and hairy vetch (Vicia villosa Roth) were established as whole plots along with a winter fallow area. Split plot treatments consisted of O, N, P, K, NP, NK, and NPK starter fertilizers. The cotton (Gossypium hirsutum L.) was planted with a conservation tillage planting unit with in‐row subsoilers. The starter fertilizers were applied deep (8 to 10 inches) in the subsoil track. Greenhouse studies were also conducted with soil from whole plot areas top dressed with corresponding legume tissue at a rate of .9 g tissue/500 g soil. Seedlings in the greenhouse were rated for disease and emergence, and dry weights were recorded.

Cotton populations in field studies were lower in legume mulched than fallow soils in 1984. Application of starter fertilizers generally increased harvest populations, particularly the NK combination. In 1983, cotton growth was greater in vetch than other soils, but responses to starter fertilizers varied with analyses and years. Seed cotton yields were consistently high with P starter, although P did not always improve cotton stands and growth. When averaged across years and cover crops, yields were 3151, 3031, 2865, 2790, 2753, 2741, 2512, and 2364 for P, NP, P, NP, K, NPK, N and O, starter treatments respectively.

Greenhouse studies indicated that starter fertilizer improved cotton emergence in legume soils, but decreased emergence in fallow soils. Disease ratings of emerged seedlings were more severe when starter fertilizer was used than when it was not used. Thus, starter fertilizer increased emergence and survival, despite high disease ratings. Cotton seedling growth generally increased when poor emergence reduced cotton seedling competition.     

Differences in growth and yield in response to cadmium toxicity in cotton genotypes

A greenhouse hydroponic experiment was conducted to study the effects of cadmium (Cd; 0, 0.1, 1.0, 10 μM in nutrient solution) on yield and yield components as well as Cd concentration and accumulation in three cotton genotypes (Simian 3, Zhongmian 16, Zhongmian 16–2). The results showed that Cd concentration in different organs increased with increasing Cd levels in the nutrient solution in the following order: root > petiole > xylem > fruiting branch, leaf > phloem in vegetative organs and seed coat, seed nut > boll shell > fiber in reproductive organs. There were significant genotypic differences in functional leaf and petiole Cd concentrations at 1 and 10 μM Cd treatments, with the cultivar Simian 3 showing higher Cd concentrations and greater reductions in lint yield than the other two genotypes.     

硫肥对棉花蕾铃脱落及产量的影响

本文就硫素与棉花蕾铃脱落和产量的关系进行了研究。试验结果表明,在传统施肥量330kg N/hm2,60kg P/hm2情况下,棉花蕾铃脱落率达70.3%;在增加氮磷肥施用量并补施硫酸钾情况下,棉花蕾铃脱落率为68.5%,说明硫酸钾有效降低了棉花的蕾铃脱落率。在传统施肥量基础上施用硫磺30.0kg/hm2对棉花的蕾铃脱落没有影响,但是增加了棉花单株结铃数、提高了产量。与不施硫相比,施硫使棉花单株结铃数增加14.7%,产量增加了15.7%。上述结果说明,在新疆高产棉田中使用单质硫肥能够进一步增加棉花产量。