低温处理对小麦几丁质酶活性的影响

[目的]研究低温对小麦几丁质酶基因的影响。[方法]对120个小麦品种进行低温处理,测定处理前后的小麦几丁质酶活力,分析低温对小麦几丁质酶活性的影响。[结果]常温下45个小麦品种的几丁质酶活性在0.5～0.8,42个小麦品种的几丁质酶活性在0.8～1.1,几丁质酶活性小于0.5和大于1.1的小麦品种仅占所测定小麦品种总量的27.5%。低温培养后所有品种的几丁质酶活性均有不同程度的提高。小麦几丁质酶活性的升高范围为0～1,74个小麦品种的几丁质酶活性升高了0～0.2,占所测小麦品种总量的61.7%;37个小麦品种的几丁质酶活性升高了0.2～0.4,仅9个小麦品种的几丁质酶活性升高了0.4以上,占所测小麦品种总量的7.5%。[结论]低温条件诱导了小麦中的几丁质酶基因表达,从而抵抗不利环境。     

蚕蛹壳几丁质降解菌的分离筛选及其产酶条件优化

[目的]对产几丁质酶菌株发酵产酶活性工艺进行优化。[方法]将筛选得到的1株产几丁质酶菌株G-254进行驯化培养,通过单因素试验考察了不同碳源、氮源和无机盐对菌株产酶活的影响;进行响应面试验,以菌株发酵所产酶活为响应值,确定最佳的发酵产酶工艺条件。[结果]菌株G-254发酵产几丁质酶最佳发酵条件为,葡萄糖8%,牛肉膏料5%,硫酸镁0.07%,在此条件下获得的几丁质酶活为6.86 U。[结论]提高了菌株发酵产几丁质酶酶活,为后续工业化发酵生产奠定了基础。     

栝蒌产几丁质酶菌株的多样性研究

[目的]探讨栝蒌产几丁质酶菌种的多样性。[方法]以绿色（未成熟）和黄色（已成熟）的栝蒌为材料,均以透明圈法筛选其产几丁质酶的菌株,并以DNS法测定酶活。[结果]从绿色果皮、绿色果肉、红色果皮、红色果肉中分别筛选得菌株91、6、71、4株,活性最高的是从绿色果皮中筛得的LP-1,其活性为0.450 U。[结论]该研究为栝蒌的开发和利用提供了理论依据,同时拓宽了高产几丁质酶菌种筛选材料选择的途径。     

生防菌BC2007对番茄促生防病作用的研究

[目的]探讨生防菌BC2007对番茄的促生防病作用。[方法]对重组的基因工程菌BC2007的GFP基因以及苯酚羟化酶基因进行了检测,并通过盆栽试验比较了BC2001和BC2007对番茄的促生防病作用。[结果]生防菌BC2007能减少番茄根系线虫瘤并对番茄的营养生长具有促进作用,也有利于后期产量的形成。[结论]为生物杀线虫菌剂的研制提了供理论依据。     

羊草ClassⅡ几丁质酶基因的克隆及序列分析

1材料与方法 1．1植物材料采摘自然生长于黑龙江省安达市（119°28’E,44°1’N）盐碱地的羊草叶片,-70℃冰箱保存。1．2总RNA的分离与eDNA文库的构建用液氮研磨叶片后,用TRIZOL Reagent（GIBCO BRL）提取总RNA,使用PolyATtract mRNA Isolation Systems（Promega）提取mRNA。然后采用Stratagene提供的cDNA合成体系合成cDNA,体外包装和扩增,构建羊草叶片的cDNA文库。     

Isolation and characterisation of a xylanase inhibitor Xip-II gene from durum wheat

Cereals contain xylanase inhibitor proteins (XIPs) which inhibit microbial xylanases from glycoside hydrolase families 10 and 11. Here, we report for the first time the isolation and characterisation of a genomic clone containing a xylanase inhibitor gene. This gene, Xip-II, isolated from a durum wheat genomic library (Triticum durum Desf.) encodes a mature protein of 307 amino acid (aa) residues that shares highest aa sequence identity (64%) with the rice RIXI xylanase inhibitor. XIP-II showed inhibition against family 11 xylanases and no chitinase activity. In silico analysis of the 5′ promoter region of Xip-II revealed sequences with similarity to known cis regulatory elements upstream from the initiation codon. In particular, the identification of a number of cis-acting elements controlling the expression of defence and seed-specific genes supports the role for this class of inhibitors in plant defence against pathogens but also provides new clues on a potential role in plant development.     

高产几丁质酶的粘质沙雷氏菌株的诱变选育

以黑龙江省科学院微生物研究所实验室保存的1株粘质沙雷氏菌S68为出发菌株,进行原生质体紫外线、Na NO2和复合诱变,通过透明圈对比和酶活测定,最终获得1株产酶量高于原始菌株4.3倍的粘质沙雷氏菌诱变菌株,产酶量达到4.98μg·h-1。确定该菌株最佳诱变方法为复合诱变,首先进行紫外诱变(高度为30 cm,诱变时间为12 s),再用化学诱变(0.4 mol·L-1Na NO2诱变5 min),经验证该诱变菌株遗传稳定。     

两个棉花几丁质酶基因的克隆与表达分析

 从棉花黄萎病缩减杂交库中得到了分属Ⅲ型和Ⅳ型几丁质酶的两个片段,它们都具有完整的3’末端。通过RACE与RT-PCR结合获得了这2个基因完整的读码框序列。其中Ⅳ型几丁质酶的开放读码框为678 bp,编码长为226个氨基酸的蛋白,命名为Ghachi4;Ⅲ型几丁质酶的全长为1390 bp,编码长为298个氨基酸的蛋白,命名为Ghachi3。它们与拟南芥同源基因的相似性分别达到65%和71%。利用网上分析软件发现这2个基因都有信号肽序列,并预测编码的蛋白位于胞质体外。半定量RT PCR发现Ghachi3在花、蕾和韧皮部中的表达量较高,而Ghachi4在韧皮部表达量最高。Ghachi4仅在抗病品种受黄萎病和枯萎病的诱导后表达上调,而Ghachi3还在感病品种中受黄萎病的诱导表达。同时,在常抗棉中2个基因的表达都受到ABA的强烈诱导。推测这2个基因可能参与生物胁迫或非生物胁迫的防御反应。     

普通小麦几丁质酶基因的克隆与表达分析

几丁质酶是植物重要的防卫因子之一,与植物抗病性密切相关。为深入了解几丁质酶基因表达及功能,先利用引物ChiF/ChiR从10份具有不同抗病性的小麦中克隆出几丁质酶基因后对其进行序列分析及原核表达分析,再利用荧光定量PCR(qRT-PCR)技术分析小麦根、茎和叶不同组织中几丁质酶基因的表达情况,并选取在小麦根、茎和叶中表达量最高的几丁质酶基因进行真菌性病害的抗性反应试验。结果表明,从10份具有不同抗病性的小麦中克隆得到5条长度约960bp的几丁质酶基因序列,其中1条序列与已公布的Chi-3基因序列(序列登录号为KJ507387)一致,其余序列命名为Cht1~Cht4(GenBank登录号为KR049247~KR049250)。序列分析表明,克隆所得序列无内含子,编码的几丁质酶属于ClassⅠb类型,也属于糖苷水解酶第19家族,是胞外分泌蛋白且兼具溶菌酶活性。SDS-PAGE分析表明,重组质粒pE-Chi能在BL21(DE3)中表达一个33kDa左右的特异蛋白。qRT-PCR分析表明,这5种几丁质酶基因在小麦根、茎和叶中均有表达,但在不同器官中表达量差异较大,其中,叶中表达量最高,根中次之,茎中最低;同时,这5种几丁质酶基因在同一器官中表达量大小具体表现为Cht4Cht2Cht3Cht1Chi-3。选取相对表达量最高的Cht4基因验证其对真菌性病害的抗性反应,结果表明,在条锈菌胁迫下,Cht4基因在抗病材料92R137中的表达量显著高于感病材料铭贤169,并且均高于同等条件下接ddH2O的表达量。本研究扩增到的几丁质酶基因在小麦中组成型表达,说明其参与了小麦的正常生长发育过程;Cht4基因的表达受条锈菌诱导,推测其可能参与了小麦叶部抗条锈菌的防御反应。     

Isolation and characterization of chitinolytic rhizobacteria for the management of Fusarium wilt in tomato

Sixty-three chitinase producing rhizobacteria (CRB) were isolated from 57 rhizospheric soil samples of tomato (Lycopersicon esculentum Mill.) growing in different regions of Karnataka, India. Among these, 13 CRB isolates were selected based on their ability to produce chitinase, colonize roots of tomato seedlings and reduce Fusarium wilt incidence. Four of these isolates produced statistically higher levels of chitinase and also zone of clearance/colony size (CZ/CS) ratios. One Bacillus subtilis isolate (CRB20) substantially reduced the severity of Fusarium wilt under greenhouse conditions. Combined application of chitin or crude fungal cell wall (CFCW) along with this isolate, substantially enhanced the ability of the isolate to colonize tomato roots and reduced the severity of Fusarium wilt. Under greenhouse conditions, amendments of chitin and CFCW along with isolate CRB20 significantly enhanced plant height, fresh weight, number of fruits per plant and average weight of fruit compared to the untreated control. The study clearly established the significance of CRB isolates and chitin/CFCW amendment in promoting plant growth and suppression of Fusarium oxysporum, and indicated the possibility of their use for Fusarium wilt management in tomato cultivation.