【作者】 李郑义；

【导师】 安千里；

【作者基本信息】 浙江大学 ， 植物病理学， 2011， 硕士

【摘要】 植物在遭受胁迫时产生乙烯。高浓度乙烯抑制植物生长。具1-氨基环丙烷-1羧酸(ACC)脱氨酶的细菌能通过降解乙烯合成前体ACC而降低植物产生乙烯的水平。在恶化的农田生态下,接种具ACC脱氨酶的细菌往往比接种具其他促植物生长特性的细菌,如固氮菌,更易产生促植物生长的效应。于是,本研究致力于从固氮菌和能在无氮培养基上生长的细菌中筛选具ACC脱氨酶的细菌并应用于农作物生产。分离具ACC脱氨酶细菌通用的方法是用以ACC为唯一氮源的基本培养基来筛选,但对于能在无氮培养基上生长的细菌,这种筛选方法显然不够灵敏。而对于从大量能在无氮培养基上生长的细菌中筛选具ACC脱氨酶的细菌则需要新的高通量筛选方法。本研究以乙二醇为溶剂加入抗坏血酸配制茚三酮试剂,在沸水浴上加热聚丙烯96孔烟囱型PCR板中的反应液,测定反应后液体在570 nm处的吸光值计算ACC的浓度,建立了高通量的茚三酮-ACC比色法用于测定细菌对ACC的消耗。接着,以43个从甘蔗分离的固氮菌和68个从玉米分离的能在无氮培养基上生长的菌株为材料,快速筛选到6个能消耗ACC的细菌,用2,4-二硝基苯肼比色法检测证实这6个菌株都具有ACC脱氨酶活性。因此,高通量茚三酮—ACC比色法适合高通量筛选具ACC脱氨酶活性细菌。用该法进一步从153个从甘蔗分离的能在无氮培养基上生长的菌株和268个从水稻或东南景天分离的能在以ACC为唯一氮源的基本培养基上生长的菌株中筛选出52个具ACC脱氨酶活性的细菌。在分子水平鉴定具ACC脱氨酶的细菌是通过用简并引物扩增ACC脱氨酶结构基因acdS或用DNA杂交检测acdS。已发表的研究往往参考少数菌株的acdS序列设计了简并引物,扩增特异性差,还不如菌落杂交有效；而菌落杂交也需要针对不同类型的acdS用相应类型的探针,探针的广谱性弱。本研究根据ACC脱氨酶的蛋白结构、氨基酸保守性分析及其与同源蛋白D-半胱氨酸脱巯基酶在结构上的区别,用CODEHOP法则设计了广谱特异的acdS简并引物：CodehopACCf1、CodehopACCf2、CodehopACCf3和CodehopACCr,并成功扩增得到所有58株具ACC脱氨酶细菌的acdS基因片段,实现了在分子水平高通量鉴定具ACC脱氨酶的细菌。在这58株细菌的acdS基因片段中,有一些归于新的acdS类群。而且,通过构建所有已知具有ACC脱氨酶活性细菌的acdS和相应16S rDNA的系统进化树揭示了acdS存在广泛的基因水平转移现象。本研究还对从玉米和水稻分离筛选的43株具ACC脱氨酶细菌的多种促植物生长特性包括分泌IAA、固氮、溶磷、分泌嗜铁素和拮抗病原真菌做了分析,发现这些细菌还具有一种以上其他促植物生长的特性,显示出促植物生长的应用潜能。其中16株伯克氏菌不仅能解磷和分泌嗜铁素,而且具有广谱的拮抗病原真菌的能力,但这些伯克氏菌在亲缘关系上与包含机会致病菌的洋葱伯克氏菌群接近。虽没有从这些伯克氏菌中检测到BCESM致病因子,但多数菌株对洋葱有害。基因水平转移acdS有可能使病原菌具有了ACC脱氨酶。因此,在应用具ACC脱氨酶活性细菌促植物生长时,要严格检测菌株可能存在的致病性。更多还原

【Abstract】 When plants are exposed to stressful conditions, they often respond by producing ethylene. High level of ethylene inhibits plant growth. Plant growth-promoting bacteria that contain the enzyme 1-aminocyclopropane-l-carboxylate (ACC) deaminase can catalyze the cleavage of ACC, the immediate precursor of ethylene in plants, and thus lower the ethylene levels. Under current deteriorated field ecosystems in China, inoculation of bacteria containing ACC-deaminase often leads to a better plant growth than inoculation of some other plant growth-promoting bacteria, such as nitrogen-fixing bacteria. Hence, this study is focused on screening the bacteria containing ACC-deaminase from bacteria that are able to fix N2 or grow on nitrogen-free media and use them to promote crop production.Selective minimal media supplying ACC as the sole nitrogen source are widely used to isolate bacteria containing ACC-deaminase. However, this isolation method is not sensitive for bacteria that are able to grow on nitrogen-free media. And a high-throughput method is required to screen bacteria containing ACC deamianse from a large number of bacterial isolates that are able to grow on nitrogen-free media. This study developed a high-throughput colorimetric method based on ninhydrin reaction with ACC to measure bacterial consumption of ACC. In this method, ethylene glycol was used as the solvent to stabilize the ninhydrin regent and color development, ascorbic acid was used as the reducing regent to prevent the oxidation of the hydrindantin, and polypropylene chimney-top 96-well PCR plates were heated on boiling water bath, ACC concentration was determined by measurement of the absorbance at 570nm. By using this method six bacterial isolates consuming ACC were rapidly screened out from 43 nitrogen-fixing bacteria isolated from sugarcane and 68 bacterial isolates that were able to grow on nitrogen-free media and were isolated from maize. And the six isolates were demonstrated to possess ACC deaminase activity by using the colorimetric 2,4-dinitrophenyl-hydrazine assay. Therefore, the 96-well plate ninhydrin-ACC assay enables a high-throughput screening of bacteria containing ACC deaminase from large numbers of isolates. Furthermore, other 52 isolates containing ACC deaminase were screened out from 153 isolates that were able to grow on nitrogen-free medium and were isolated from sugarcane, and 268 isolates that were able to grow on minimal media containing ACC as the sole nitrogen source and were isolated from rice or Sedum alfredii.Molecular identification of bacteria containing ACC deaminase usually uses PCR amplification or DNA hybridization of the ACC deaminase structural gene (acdS). Previous studies designed degenerative primers based on a few available sequences. PCR amplification based on these primers was not as efficient as hybridization to detect acdS and yielded nonspecific amplicons whereas hybridization of different groups of acdS required different probes. This study designed broad-spectrum specific degenerative primers of CodehopACCf1、CodehopACCf2、CodehopACCf3 and CodehopACCr to amplify acdS based on the protein structures of ACC deaminases, differentiation of the conserved amino acids between ACC deaminase and D-cysteine desulfhydrase, and the CODEHOP strategy. PCR amplification based on the CODEHOP primers successfully amplified acdS from all the 58 bacterial isolates containing ACC deaminase and thus accomplished a high-throughput molecular method to identify bacteria containing ACC deaminase. Phylogenetic analysis of acdS showed that some acdS sequences obtained in this study were grouped into new acdS groups. And phylogenetic analysis of acdS and corresponding bacterial 16S rDNA revealed a broad horizontal gene transfer of acdS.This study further determined other plant growth-promoting traits including IAA secretion, nitrogen fixation, phosphate solubilization, siderophore secretion, and pathogen antagonism for 43 isolates isolated from maize and rice. They all showed more than one of these traits. Among them,16 isolates belonged to the genus Burkholderia were able to solubilize phosphate, secrete siderophore, and antagonize broad pathogenic fungi. Although no Burkholderia cepacia epidemic strain marker was detected from these isolates, they were close related to opportunistic pathogens Burkholderia cepacia complex and most of them were pathogenic to onion bulb. Pathogens may obtain ACC deaminase via horizontal transfer of acdS. Therefore, application of potential plant growth-promoting bacteria containing ACC deaminase requires strict pathogenic test.更多还原