广西甘蔗野生种质内生细菌资源及其辅助甘蔗耐瘠抗旱的基础研究

Increase of nitrogen and water utilization efficiency and breeding of new infertility- and drought-tolerant, high-sugar and high-yield cultivars are the keys to ensure the sustainable development of sugarcane and sugar production in China. Wild Saccharum spontaneum and Erianthus arundinaceum plants are important infertility- and drought-tolerant germplasms for sugarcane breeding. Endophytic diazotrophs can help wild sugarcane germplasms to tolerate infertility via biological nitrogen fixation. ACC deaminase-containing bacteria can help plants to tolerate stress via degradation of ACC to reduce ethylene production. We planned to sample the roots and stems from wild Saccharum spontaneum and Erianthus arundinaceum plants grown in infertile and drought sloping fields in Guangxi and amplify bacterial nifH and acdS transcripts and analyze the active nitrogen-fixing and ACC-degrading bacterial community in the wild germplasms. We will also isolate culturable nitrogen-fixing bacteria and ACC deaminase-containing bacteria and amplify their nifH or acdS genes and then find the active nitrogen-fixing bacteria and ACC-degrading bacteria guided by the transcriptomic analyses and sequence comparison. We will further inoculate micropropagated plantlets of intolerant sugarcane cultivars with the selected nitrogen-fixing bacteria or ACC-degrading bacteria and investigate their associative nitrogen fixation and assistance to drought tolerance. By taken advantage of an ACC deaminase-containing bacterial strain GT21-15 isolated from a drought-tolerant sugarcane cultivar, we will construct an acdS mutant strain and demonstrate whether the ACC deaminase plays a role in assistance to sugarcane cultivars to tolerate drought stress. This study will provide bacterial resources and application evidence to increase infertility- and drought-tolerance for sugarcane cultivars in China.

提高甘蔗的氮肥和水分利用效率、培育耐瘠抗旱且高产高糖的新品种是保障我国甘蔗糖业可持续发展的关键。野生割手密和斑茅是甘蔗育种的重要耐瘠抗旱种质资源。内生固氮菌能通过生物固氮帮助甘蔗野生种质耐瘠。有ACC脱氨酶的细菌能通过降解ACC而降低植物产生胁迫乙烯的水平而帮助植物抗逆。本项目拟从广西贫瘠旱坡地采集野生割手密和斑茅的根和茎，用RT-PCR从中扩增细菌nifH和acdS的转录组，测序分析其中活跃固氮或降解ACC细菌的群落组成，同时从中分离可培养的固氮菌和有ACC脱氨酶的细菌，选出与转录组学分析相符的细菌，进而接种不耐瘠或不抗旱甘蔗栽培品种的组培苗，检测其联合固氮或辅助抗旱的能力；并利用已有的GT21-15菌株构建ACC脱氨酶失活突变体来检验有ACC脱氨酶的细菌能否通过ACC脱氨酶的作用帮助甘蔗抗旱，为提高我国栽培甘蔗的耐瘠抗旱能力开发细菌资源并提供实践依据。

野生割手密和斑茅是重要的甘蔗育种的耐瘠抗旱种质资源。内生固氮菌能通过生物固氮帮助甘蔗野生种质耐瘠。有ACC脱氨酶的细菌能通过降解ACC而降低植物产生胁迫乙烯的水平而帮助植物抗逆。本项目的实施证实了基于扩增内生细菌固氮酶基因nifH和ACC脱氨酶基因acdS转录组，指导分离筛选野生甘蔗种质体内功能固氮菌和有ACC脱氨酶细菌的可行性和有效性，获得了帮助甘蔗耐瘠抗旱的内生细菌及其基因组信息，实现了部分预期目标，为研究和利用内生细菌辅助甘蔗耐瘠抗旱奠定了基础。.具体研究内容和结果如下。.1）从广西崇左和来宾地区的贫瘠旱坡地采集野生割手密和斑茅的根和茎，用RT-PCR从中扩增细菌nifH和acdS的转录组，通过克隆测序发现割手密和斑茅体内有根瘤菌属和慢生根瘤菌属的细菌在表达nifH，有中华根瘤菌属/剑菌属的细菌在表达acdS。.2）从割手密和斑茅的根和茎中分离到390个细菌分离物，通过扩增分析细菌的16S rRNA基因序列，已鉴定了220个细菌的归属。通过扩增nifH筛选到26个固氮菌，扩增acdS筛选到17个有ACC脱氨酶的细菌。.3）从割手密根中分离到根瘤菌属细菌CZGRD1。它的nifH序列与一个在割手密根中表达的nifH序列相同，它可能是与野生割手密联合固氮的功能固氮菌。用CZGRD1接种新台糖22号甘蔗组培苗，证实CZGRD1能与甘蔗联合固氮并显著促进甘蔗生长。.4）从斑茅根中分离到有ACC脱氨酶的剑菌属细菌WXBRA3。它的acdS序列与一个在斑茅根中表达的acdS序列高度相似，它可能在斑茅体内发挥ACC脱氨酶的作用。用WXBRA3接种新台糖22号甘蔗苗，证实WXBRA3能促进甘蔗生长并帮助甘蔗抗旱。.5）用从耐瘠抗旱先锋植物沙鞭的根中分离得到的固氮菌Klebsiella oxytoca菌株SA2接种新台糖22号甘蔗组培苗，证实SA2能与甘蔗联合固氮并显著促进甘蔗生长。用共聚焦显微术发现gfp标记的SA2能在甘蔗根皮层和根毛内定殖。.6）用Illumina HiSeq2000高通量测序系统测定了WXBRA3和SA2菌株的基因组，都获得了覆盖率大于99.9%的基因组精细图。