中国农业科学
中國農業科學
중국농업과학
SCIENTIA AGRICULTURA SINICA
2015年
8期
1463-1472
,共10页
肖瑞霞%王新国%夏国军%李永春%牛洪斌%王翔%尹钧%任江萍
肖瑞霞%王新國%夏國軍%李永春%牛洪斌%王翔%尹鈞%任江萍
초서하%왕신국%하국군%리영춘%우홍빈%왕상%윤균%임강평
小麦%C2结构域蛋白%基因克隆%表达分析
小麥%C2結構域蛋白%基因剋隆%錶達分析
소맥%C2결구역단백%기인극륭%표체분석
wheat (Triticum aestivum)%C2 domain protein%gene cloning%expression analysis
【目的】克隆与逆境胁迫相关的基因，并对其序列特征、进化关系和表达特性进行分析，探讨该基因在小麦抗逆调控过程中的生物学功能，为进一步解析植物的抗逆机制提供候选基因和理论依据。【方法】以 cDNA芯片数据获得的水分胁迫诱导上调表达基因EST序列为探针，对小麦EST数据库进行搜索，筛选与探针同源性在97％以上的EST序列，通过电子克隆结合RT-PCR获得该基因cDNA全长，采用生物信息学软件分析比较克隆基因的保守结构域及序列特征；采用MEGA6.0软件构建该基因的系统进化树；将测序正确的该基因片段通过EcoRⅠ和HindⅢ限制性内切酶酶切连接至原核表达载体pMAL-c2X，重组质粒转化大肠杆菌BL21，经终浓度为0.3 mmol·L-1 IPTG诱导1—5 h后，用SDS-PAGE分析融合蛋白的表达；采用实时荧光定量PCR（qRT-PCR）分析该基因在小麦不同组织间的表达差异及其在低温、干旱、高温和ABA处理下的表达模式。【结果】成功获得小麦cDNA全长序列，命名为TaC2DP1。该基因序列全长为1356 bp，包含一个1209 bp的开放阅读框（ORF），5′端非编码区50 bp，3′端非编码区97 bp，编码402个氨基酸，推导编码蛋白质的预测分子量为43.41 kD，等电点为4.30，属于酸性蛋白，BLAST分析表明，该蛋白含有一个与钙离子结合的结构域，称为C2结构域（C2-domain）。多序列比对及进化树分析表明，TaC2DP1与乌拉尔图小麦TuC2亲缘关系最近，二者具有高度的同源性，其编码的氨基酸一致性达到91%；蛋白质结构预测分析显示TaC2DP1无跨膜螺旋和双硫键，亚细胞定位于细胞质中；成功构建了该基因的原核表达载体pMAL-c2X-TaC2DP1，在IPTG诱导下得到90 kD左右的蛋白，与理论值一致。通过实时荧光定量PCR进行TaC2DP1表达分析，显示该基因在小麦的根、茎、叶、幼穗、未成熟籽粒、胚及胚乳中均有表达，其中在幼穗中表达量最高，在花后5 d籽粒中表达量最低。TaC2DP1可被植物激素ABA诱导而上调表达；干旱胁迫过程中， TaC2DP1受胁迫诱导呈稳定上调表达趋势；高温和低温胁迫过程中，TaC2DP1均在胁迫后的0.5 h迅速诱导上调表达，分别为对照的21和17倍。推测该基因可能参与小麦ABA 信号通路中对逆境胁迫的抗性反应。【结论】获得小麦TaC2DP1的全长序列，其编码蛋白含有与钙离子结合的C2结构域；在低温、干旱、高温和ABA逆境胁迫下， TaC2DP1属于依赖于ABA胁迫响应基因调控网络，可能在干旱、低温和热胁迫中发挥重要作用。
【目的】剋隆與逆境脅迫相關的基因，併對其序列特徵、進化關繫和錶達特性進行分析，探討該基因在小麥抗逆調控過程中的生物學功能，為進一步解析植物的抗逆機製提供候選基因和理論依據。【方法】以 cDNA芯片數據穫得的水分脅迫誘導上調錶達基因EST序列為探針，對小麥EST數據庫進行搜索，篩選與探針同源性在97％以上的EST序列，通過電子剋隆結閤RT-PCR穫得該基因cDNA全長，採用生物信息學軟件分析比較剋隆基因的保守結構域及序列特徵；採用MEGA6.0軟件構建該基因的繫統進化樹；將測序正確的該基因片段通過EcoRⅠ和HindⅢ限製性內切酶酶切連接至原覈錶達載體pMAL-c2X，重組質粒轉化大腸桿菌BL21，經終濃度為0.3 mmol·L-1 IPTG誘導1—5 h後，用SDS-PAGE分析融閤蛋白的錶達；採用實時熒光定量PCR（qRT-PCR）分析該基因在小麥不同組織間的錶達差異及其在低溫、榦旱、高溫和ABA處理下的錶達模式。【結果】成功穫得小麥cDNA全長序列，命名為TaC2DP1。該基因序列全長為1356 bp，包含一箇1209 bp的開放閱讀框（ORF），5′耑非編碼區50 bp，3′耑非編碼區97 bp，編碼402箇氨基痠，推導編碼蛋白質的預測分子量為43.41 kD，等電點為4.30，屬于痠性蛋白，BLAST分析錶明，該蛋白含有一箇與鈣離子結閤的結構域，稱為C2結構域（C2-domain）。多序列比對及進化樹分析錶明，TaC2DP1與烏拉爾圖小麥TuC2親緣關繫最近，二者具有高度的同源性，其編碼的氨基痠一緻性達到91%；蛋白質結構預測分析顯示TaC2DP1無跨膜螺鏇和雙硫鍵，亞細胞定位于細胞質中；成功構建瞭該基因的原覈錶達載體pMAL-c2X-TaC2DP1，在IPTG誘導下得到90 kD左右的蛋白，與理論值一緻。通過實時熒光定量PCR進行TaC2DP1錶達分析，顯示該基因在小麥的根、莖、葉、幼穗、未成熟籽粒、胚及胚乳中均有錶達，其中在幼穗中錶達量最高，在花後5 d籽粒中錶達量最低。TaC2DP1可被植物激素ABA誘導而上調錶達；榦旱脅迫過程中， TaC2DP1受脅迫誘導呈穩定上調錶達趨勢；高溫和低溫脅迫過程中，TaC2DP1均在脅迫後的0.5 h迅速誘導上調錶達，分彆為對照的21和17倍。推測該基因可能參與小麥ABA 信號通路中對逆境脅迫的抗性反應。【結論】穫得小麥TaC2DP1的全長序列，其編碼蛋白含有與鈣離子結閤的C2結構域；在低溫、榦旱、高溫和ABA逆境脅迫下， TaC2DP1屬于依賴于ABA脅迫響應基因調控網絡，可能在榦旱、低溫和熱脅迫中髮揮重要作用。
【목적】극륭여역경협박상관적기인，병대기서렬특정、진화관계화표체특성진행분석，탐토해기인재소맥항역조공과정중적생물학공능，위진일보해석식물적항역궤제제공후선기인화이론의거。【방법】이 cDNA심편수거획득적수분협박유도상조표체기인EST서렬위탐침，대소맥EST수거고진행수색，사선여탐침동원성재97％이상적EST서렬，통과전자극륭결합RT-PCR획득해기인cDNA전장，채용생물신식학연건분석비교극륭기인적보수결구역급서렬특정；채용MEGA6.0연건구건해기인적계통진화수；장측서정학적해기인편단통과EcoRⅠ화HindⅢ한제성내절매매절련접지원핵표체재체pMAL-c2X，중조질립전화대장간균BL21，경종농도위0.3 mmol·L-1 IPTG유도1—5 h후，용SDS-PAGE분석융합단백적표체；채용실시형광정량PCR（qRT-PCR）분석해기인재소맥불동조직간적표체차이급기재저온、간한、고온화ABA처리하적표체모식。【결과】성공획득소맥cDNA전장서렬，명명위TaC2DP1。해기인서렬전장위1356 bp，포함일개1209 bp적개방열독광（ORF），5′단비편마구50 bp，3′단비편마구97 bp，편마402개안기산，추도편마단백질적예측분자량위43.41 kD，등전점위4.30，속우산성단백，BLAST분석표명，해단백함유일개여개리자결합적결구역，칭위C2결구역（C2-domain）。다서렬비대급진화수분석표명，TaC2DP1여오랍이도소맥TuC2친연관계최근，이자구유고도적동원성，기편마적안기산일치성체도91%；단백질결구예측분석현시TaC2DP1무과막라선화쌍류건，아세포정위우세포질중；성공구건료해기인적원핵표체재체pMAL-c2X-TaC2DP1，재IPTG유도하득도90 kD좌우적단백，여이론치일치。통과실시형광정량PCR진행TaC2DP1표체분석，현시해기인재소맥적근、경、협、유수、미성숙자립、배급배유중균유표체，기중재유수중표체량최고，재화후5 d자립중표체량최저。TaC2DP1가피식물격소ABA유도이상조표체；간한협박과정중， TaC2DP1수협박유도정은정상조표체추세；고온화저온협박과정중，TaC2DP1균재협박후적0.5 h신속유도상조표체，분별위대조적21화17배。추측해기인가능삼여소맥ABA 신호통로중대역경협박적항성반응。【결론】획득소맥TaC2DP1적전장서렬，기편마단백함유여개리자결합적C2결구역；재저온、간한、고온화ABA역경협박하， TaC2DP1속우의뢰우ABA협박향응기인조공망락，가능재간한、저온화열협박중발휘중요작용。
[Objective]The objective of this study is to clone the stress resistance-related gene, analyze its sequence features, evolutionary relationships and expression characteristics, investigate its biological function during the stress tolerance of wheat, and to provide candidate gene and a theoretical foundation for clarifying molecular mechanism of stress resistance.[Method]Using an up-regulated EST obtained by cDNA chip as a probe to search the wheat EST databases, filter out the ESTs sequences with the homology of 97%of the probe, a full-length cDNA sequence was cloned from wheat by in silico cloning and reverse transcription PCR (RT-PCR) method. The conserved domains and sequence features of the gene were analyzed by bioinformatics’ methods. A phylogenetic tree was constructed using the MEGA 6.0 software, and then the cloned gene ORF was inserted into the expression vector pMAL-c2X by EcoR I and Hind Ⅲ digestion. The recombinant plasmid was transformed into E. coli BL21 and expressed under the induction with 0.3 mmol·L-1 IPTG for 1-5 h. The expression of the fusion protein was detected by SDS-PAGE. The expression profiles of the cloned gene in various tissues and in response to cold, drought, heat and abscisic acid (ABA) treatment were investigated using quantitative real-time PCR (qRT-PCR).[Result]The full-length cDNA sequence designated as TaC2DP1 from wheat is 1 356 bp in length, contains a 1 209 bp open reading frame (ORF), with 50 bp in the 5' UTR and 97 bp in the 3' UTR. TaC2DP1 was predicted to encode a 402 amino acid protein with a molecular mass of 43.41 kD and isoelectric point of 4.30, it belongs to the acidic protein. BLAST analysis revealed that the protein contains a C2-domain and was predicted to be a Ca2+binding domain. Multiple sequence alignment and phylogenetic tree analysis showed that TaC2DP1 had the closest evolutionary relationship with a C2-domain protein in Triticum urartu with unknown function, and shares 91% identity in amino acids. Protein structure prediction showed that TaC2DP1 had not transmembrane helix and disulfide bond and might be localized in cytoplasm. The prokaryotic expression vector of TaC2DP1, pMAL-c2X-TaC2DP1, was successfully constructed. The expression of fusion protein was obtained by inducing with IPTG and its relative molecular weight was 90 kD, which was consistent with the theoretical value. Real time quantitative PCR (qRT-PCR) analysis revealed that TaC2DP1 constitutively expressed in all tested roots, stems, leaves, immature ears, immature seeds, embryo and endosperm, the expression level of TaC2DP1 in immature ears was the highest, while its expression level was very low in seeds at 5 days after anthesis (DAA). The expression profiling revealed that TaC2DP1 was induced by plant hormone ABA and the expression of TaC2DP1 was steadily up-regulated in any of the time point under drought stress. TaC2DP1 was rapidly up-regulated within 0.5 h of cold and heat stress treatments and the expression level was 21 and 17 times than those of control, respectively. These results revealed that TaC2DP1 might be involved in stress resistance-related response of ABA signaling pathways in wheat leaves.[Conclusion]A full-length cDNA of TaC2DP1 was isolated and the typical a Ca2+ binding domain was found in the deduced proteins. The expression of TaC2DP1 was all up-regulated under drought, high and low temperature and ABA treatments, showing that TaC2DP1 was involved in the ABA dependent stress-responding gene network. It was deduced that TaC2DP1 might play an important regulation role under stress in wheat.
