CAJ | 학술논문

【目的】研究川桑液泡膜型 Na +/H +逆向转运蛋白( NHX)基因的功能，探究桑树耐盐机制，为植物抗逆基因工程筛选提供优良的候选基因。【方法】以川桑基因组数据库为基础，基于同源基因序列的保守性，以川桑叶片 cDNA为模板克隆川桑液泡膜型 NHX基因；利用生物学软件和在线公共平台，分析所得基因编码的蛋白质序列及功能结构域，并构建系统进化树，分析与其他物种的亲缘关系。采用荧光定量 PCR方法研究在 NaCl胁迫条件下不同时间段‘湖桑32号’根、茎、叶等不同组织中桑树 NHX1表达量的变化情况；通过构建超量表达载体，将其转化到拟南芥中，分析转基因拟南芥在 NaCl胁迫环境中的种子发芽数，根长、侧根生长情况和幼苗成活率，并对转基因拟南芥连续浇灌含高浓度 NaCl的营养液，研究过量表达 NHX 基因对拟南芥的影响。【结果】本研究得到1个液泡膜型 NHX 基因，命名为 MnNHX1( GenBank 登录号： KJ720637)；该基因 ORF 长度为1644 bp，编码547个氨基酸残基，具有 Na +/H +交换泵，且在其上游含有抑制剂氨氯吡嗪脒结合位点( LFFIYLLPPI)以及糖基化位点等结构域，TMHMM 在线程序预测 MnNHX1具有12个明显的跨膜结构区；系统进化树分析结果显示，MnNHX1具有较高的保守性，先与源于蔷薇科的桃聚合，与桑树形态学和基因组进化分析分类结果一致。荧光定量 PCR 试验表明，在无 NaCl 胁迫条件下桑树 NHX1在‘湖桑32号’根、茎、叶中均有表达；在NaCl胁迫处理12 h后，根、茎中桑树 NHX1的表达量显著增加，而后回落；而在胁迫处理24 h 后，叶中桑树 NHX1的表达量显著提高，随后回落。过量表达 MnNHX1的转基因拟南芥在 NaCl 胁迫环境中，种子发芽率低于野生型，而根长和侧根生长情况以及幼苗成活率都优于野生型；连续浇灌含高浓度 NaCl 营养液的转基因拟南芥生长状态更为优良。【结论】MnNHX1为优良的植物耐盐基因，在桑树中为组成型表达，并受NaCl胁迫诱导，表现出组织特异性。过量表达 MnNHX1的拟南芥耐盐能力显著提高，生存在盐胁迫环境中，依然具有良好的生长和发育能力。【目的】研究川桑液泡膜型 Na +/H +逆嚮轉運蛋白( NHX)基因的功能，探究桑樹耐鹽機製，為植物抗逆基因工程篩選提供優良的候選基因。【方法】以川桑基因組數據庫為基礎，基于同源基因序列的保守性，以川桑葉片 cDNA為模闆剋隆川桑液泡膜型 NHX基因；利用生物學軟件和在線公共平檯，分析所得基因編碼的蛋白質序列及功能結構域，併構建繫統進化樹，分析與其他物種的親緣關繫。採用熒光定量 PCR方法研究在 NaCl脅迫條件下不同時間段‘湖桑32號’根、莖、葉等不同組織中桑樹 NHX1錶達量的變化情況；通過構建超量錶達載體，將其轉化到擬南芥中，分析轉基因擬南芥在 NaCl脅迫環境中的種子髮芽數，根長、側根生長情況和幼苗成活率，併對轉基因擬南芥連續澆灌含高濃度 NaCl的營養液，研究過量錶達 NHX 基因對擬南芥的影響。【結果】本研究得到1箇液泡膜型 NHX 基因，命名為 MnNHX1( GenBank 登錄號： KJ720637)；該基因 ORF 長度為1644 bp，編碼547箇氨基痠殘基，具有 Na +/H +交換泵，且在其上遊含有抑製劑氨氯吡嗪脒結閤位點( LFFIYLLPPI)以及糖基化位點等結構域，TMHMM 在線程序預測 MnNHX1具有12箇明顯的跨膜結構區；繫統進化樹分析結果顯示，MnNHX1具有較高的保守性，先與源于薔薇科的桃聚閤，與桑樹形態學和基因組進化分析分類結果一緻。熒光定量 PCR 試驗錶明，在無 NaCl 脅迫條件下桑樹 NHX1在‘湖桑32號’根、莖、葉中均有錶達；在NaCl脅迫處理12 h後，根、莖中桑樹 NHX1的錶達量顯著增加，而後迴落；而在脅迫處理24 h 後，葉中桑樹 NHX1的錶達量顯著提高，隨後迴落。過量錶達 MnNHX1的轉基因擬南芥在 NaCl 脅迫環境中，種子髮芽率低于野生型，而根長和側根生長情況以及幼苗成活率都優于野生型；連續澆灌含高濃度 NaCl 營養液的轉基因擬南芥生長狀態更為優良。【結論】MnNHX1為優良的植物耐鹽基因，在桑樹中為組成型錶達，併受NaCl脅迫誘導，錶現齣組織特異性。過量錶達 MnNHX1的擬南芥耐鹽能力顯著提高，生存在鹽脅迫環境中，依然具有良好的生長和髮育能力。
【목적】연구천상액포막형 Na +/H +역향전운단백( NHX)기인적공능，탐구상수내염궤제，위식물항역기인공정사선제공우량적후선기인。【방법】이천상기인조수거고위기출，기우동원기인서렬적보수성，이천상협편 cDNA위모판극륭천상액포막형 NHX기인；이용생물학연건화재선공공평태，분석소득기인편마적단백질서렬급공능결구역，병구건계통진화수，분석여기타물충적친연관계。채용형광정량 PCR방법연구재 NaCl협박조건하불동시간단‘호상32호’근、경、협등불동조직중상수 NHX1표체량적변화정황；통과구건초량표체재체，장기전화도의남개중，분석전기인의남개재 NaCl협박배경중적충자발아수，근장、측근생장정황화유묘성활솔，병대전기인의남개련속요관함고농도 NaCl적영양액，연구과량표체 NHX 기인대의남개적영향。【결과】본연구득도1개액포막형 NHX 기인，명명위 MnNHX1( GenBank 등록호： KJ720637)；해기인 ORF 장도위1644 bp，편마547개안기산잔기，구유 Na +/H +교환빙，차재기상유함유억제제안록필진미결합위점( LFFIYLLPPI)이급당기화위점등결구역，TMHMM 재선정서예측 MnNHX1구유12개명현적과막결구구；계통진화수분석결과현시，MnNHX1구유교고적보수성，선여원우장미과적도취합，여상수형태학화기인조진화분석분류결과일치。형광정량 PCR 시험표명，재무 NaCl 협박조건하상수 NHX1재‘호상32호’근、경、협중균유표체；재NaCl협박처리12 h후，근、경중상수 NHX1적표체량현저증가，이후회락；이재협박처리24 h 후，협중상수 NHX1적표체량현저제고，수후회락。과량표체 MnNHX1적전기인의남개재 NaCl 협박배경중，충자발아솔저우야생형，이근장화측근생장정황이급유묘성활솔도우우야생형；련속요관함고농도 NaCl 영양액적전기인의남개생장상태경위우량。【결론】MnNHX1위우량적식물내염기인，재상수중위조성형표체，병수NaCl협박유도，표현출조직특이성。과량표체 MnNHX1적의남개내염능력현저제고，생존재염협박배경중，의연구유량호적생장화발육능력。
Objective] To study the function of Na + /H +antiporter ( NHX) in vacuolar membrane from mulberry tree Morus notabilis,and to explore the mechanism of salt tolerance in mulberry,and to provide an excellent candidate gene for the screening of plant resistance gene engineering. [Method]In this study,a Na + /H +antiporter gene named as MnNHX1 was identified based on the M. notabilis genomic database and other homologous sequences. The MnNHX1 was cloned using the cDNA from M. notabilis leaves as template. The analysis of the primary structure and functional domains from MnNHX1 was completed by the bioinformatics analysis. The phylogenetic tree was generated to analyse the relationships between mulberry NHX1 and other species. Quantitative PCR was conducted to analyse the expression profiles of mulberry NHX1 in different tissues of M. multicaulis‘Husang No. 32’and treatment time under NaCl stress. The overexpression vector was constructed and transformed into Arabidopsis thaliana. The seed germination rate,the growth of roots and the survival rate of seedlings of the transgenic A. thaliana were analyzed under NaCl stress. Furthermore,the transgenic A. thaliana was continuously irrigated with the nutrient solution containing high concentration of NaCl to study the functional effects of MnNHX1 gene in the transgenic A. thaliana. [Result]We cloned a Na + /H + antiporter gene designated as MnNHX1(GenBank accession No. KJ720637). The open reading frame (ORF) of MnNHX1 is 1 644 bp and encodes a protein of 547 amino acid with a Na + /H + exchange pump. At the upstream of this pump,there are some domains such as inhibitors amiloride binding sites ( LFFIYLLPPI) and glycosylation sites. The analysis of the online program of TMHMM showed that MnNHX1 have 12 obvious transmembrane region. Phylogenetic analysis showed that MnNHX1 was firstly clustered with Prunus persica from the Rosaceae family,which is consistent with morphological classification and genomic phylogenetic analysis of mulberry. Quantitative PCR showed that expression of mulberry NHX1 was detected in roots, stems and leaves of M. multicaulis‘Husang No. 32’without NaCl treatment. The expression levels of mulberry NHX1 were significantly increased followed by a drop in roots and stems after 12 h salt treatment,and in leaves after 24 h treatment. The seed germination rate of transgenetic A. thaliana overexpressed MnNHX1 was lower than that of the wild plants under the salt condition,but root length,growth of lateral roots and survival rate of seedlings were higher than those of the wild plants . When the transgenetic A. thaliana seedlings irrigated with high concentration NaCl,they grew better than the wide type plants.[Conclusion]MnNHX1 is a excellent candidate gene for improving the salt tolerance and can be constitutively expressed in mulberry tree. However,its induced expression pattern showed tissue specificity under salt condition. Furthermore,overexpression of MnNHX1 in A. thaliana can significantly improve the salt tolerance of the transgenic A. thaliana.