分子植物育种
分子植物育種
분자식물육충
MOLECULAR PLANT BREEDING
2010年
2期
370-374
,共5页
胡俊敏%张龙雨%袁正杰%张改生%韩艳芬%李亚鑫%盛英%位芳%王俊生%牛娜%马守才%李红霞
鬍俊敏%張龍雨%袁正傑%張改生%韓豔芬%李亞鑫%盛英%位芳%王俊生%牛娜%馬守纔%李紅霞
호준민%장룡우%원정걸%장개생%한염분%리아흠%성영%위방%왕준생%우나%마수재%리홍하
黏类小麦%雄性不育系%线粒体DNA%抑制性消减杂交
黏類小麥%雄性不育繫%線粒體DNA%抑製性消減雜交
점류소맥%웅성불육계%선립체DNA%억제성소감잡교
Wheat with A egilops kotschyi%Cytoplasmic male sterile lines%Mitochondrial DNA (mtDNA)%Suppres sion subtractive hybridization (SSH)
本研究利用抑制性消减杂交技术,构建了黏类小麦细胞质雄性不育线粒体DNA的消减文库.分别提取相同细胞质背景下的不育和可育等基因系线粒体基因组DNA(mtDNA),用Rsa I酶切成大小不等的片段,并各自与不同的接头连接,连续经过两次消减杂交和两次PCR扩增,将PCR产物与克隆载体连接,转化为大肠杆菌感受态细胞DH5α,经蓝白斑筛选后,再用PCR方法插入片段筛选出阳性重组质粒,构建差异DNA消减文库.在构建的不育mtDNA文库中,将SSH文库的全部扩增产物与SSH文库中的单条扩增条带分别进行胶回收和克隆转化,分别挑取54个和6个阳性克隆进行测序分析和相关功能初步比对.结果表明,不育mtDNA的SSH文库差减杂交效率较高,质量较好;回收单条扩增条带分别进行克隆测序的结果准确率较高;对测序序列进行BLASTx比对及功能注释分析发现,约90%的序列来源于线粒体基因had1和had5的非编码区.
本研究利用抑製性消減雜交技術,構建瞭黏類小麥細胞質雄性不育線粒體DNA的消減文庫.分彆提取相同細胞質揹景下的不育和可育等基因繫線粒體基因組DNA(mtDNA),用Rsa I酶切成大小不等的片段,併各自與不同的接頭連接,連續經過兩次消減雜交和兩次PCR擴增,將PCR產物與剋隆載體連接,轉化為大腸桿菌感受態細胞DH5α,經藍白斑篩選後,再用PCR方法插入片段篩選齣暘性重組質粒,構建差異DNA消減文庫.在構建的不育mtDNA文庫中,將SSH文庫的全部擴增產物與SSH文庫中的單條擴增條帶分彆進行膠迴收和剋隆轉化,分彆挑取54箇和6箇暘性剋隆進行測序分析和相關功能初步比對.結果錶明,不育mtDNA的SSH文庫差減雜交效率較高,質量較好;迴收單條擴增條帶分彆進行剋隆測序的結果準確率較高;對測序序列進行BLASTx比對及功能註釋分析髮現,約90%的序列來源于線粒體基因had1和had5的非編碼區.
본연구이용억제성소감잡교기술,구건료점류소맥세포질웅성불육선립체DNA적소감문고.분별제취상동세포질배경하적불육화가육등기인계선립체기인조DNA(mtDNA),용Rsa I매절성대소불등적편단,병각자여불동적접두련접,련속경과량차소감잡교화량차PCR확증,장PCR산물여극륭재체련접,전화위대장간균감수태세포DH5α,경람백반사선후,재용PCR방법삽입편단사선출양성중조질립,구건차이DNA소감문고.재구건적불육mtDNA문고중,장SSH문고적전부확증산물여SSH문고중적단조확증조대분별진행효회수화극륭전화,분별도취54개화6개양성극륭진행측서분석화상관공능초보비대.결과표명,불육mtDNA적SSH문고차감잡교효솔교고,질량교호;회수단조확증조대분별진행극륭측서적결과준학솔교고;대측서서렬진행BLASTx비대급공능주석분석발현,약90%적서렬래원우선립체기인had1화had5적비편마구.
In this research,we construct subtractive mtDNA library of male sterile lines by employing suppression subtractive hybridization (SSH) technology.Mitochondrial genome DNA were extracted from male sterile and fertile lines with the same cytoplasmic source.After Rsal digestion,tester DNA were divided into two groups to link with the specific adaptor 1 and adaptor 2.Then tester DNA were hybridized with driver DNA twice and underwent nested PCR twice.The PCR products were ligated with pMD19-T vector,transformed into E.coli DH5α,screened through the blue-white screening system,and then the positive recombinant plasmid were identified by PCR approach,mtDNA library was constructed successfully.54 clones from all the PCR products and 6 clones from single PCR band in the SSH library was cloned respectively,and the positive clones were sequenced and analyzed.The results showed that cDNA libraries constructed was of good quality with high efficiency of SSH,and sequencing results of recycled single PCR bands were of high accuracy.Furthermore,approximately 90% sequences analyzed were derived from non-coding regions of mitochondrial genes including nod1 and nad5 based on BLASTx and functional annotation analysis.
