小麦看家基因鉴定和水培体系磷胁迫响应基因筛选
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摘要
本研究在建立小麦水培体系的基础上,对小麦磷胁迫生理反应特征进行了系统检测,并对此体系下的小麦磷胁迫响应基因进行了初步筛选。研究还对基于mRNA检测的小麦看家基因的转录水平进行了鉴定分析。本研究为在营养严格可控的条件下,研究小麦磷胁迫反应建立了实验基础,筛选出的看家基因和磷响应基因具有进一步利用和发掘价值;研究体系和结果对发掘磷高效利用小麦基因和进行小麦种质资源的筛选都具有重要意义。研究主要结果如下:
1、采用改进的Hoagland培养基,建立并优化了小麦温室水培体系,对不同基因型小麦在水培条件下对磷饥饿胁迫的生理反应进行了研究。
(1)在建立的水培体系中,小麦植株生长正常并对磷饥饿胁迫条件做出形态建成水平的反应;半定量RT-PCR分析显示在此体系中小麦磷响应基因在转录水平上差异显著。
(2)在水培条件下,不同基因型小麦对磷饥饿胁迫的反应总体上表现在根系长度和根冠比所提高,但不同基因型小麦的反应特点有差异即在供试品种中,中国春和周麦18对磷饥饿做出的反应较为迟缓,而太空6号、豫农202和偃展4110则在较早时间对磷饥饿做出发育水平反应,随后呈现平台趋势;值得注意的是郑麦9023在对磷饥饿较早做出形态适应反应后出现明显恢复,暗示其可能存在某种显著的补偿机制。对这些反应特征的分子机理的深入分析,可为深入解析小麦代谢调控的基因网络和有效筛选磷高效小麦基因资源提供线索。
2、参考已经发表的部分拟南芥看家基因,在NCBI数据库中搜索得到10个小麦同源性较高的ESTs,通过半定量RT-PCR方法,分析了各候选基因在不同基因型小麦(偃展4110、郑麦9023)中的时空表达情况。结果表明:小麦泛肽结合酶基因(AY736121)、翻译延伸因子I-α亚基基因(M90077)和δ-液泡膜内蛋白基因(U86763)在偃展4110和郑麦9023小麦各个时期不同组织中均稳定表达,具有作为看家基因的应用潜力。
3、利用比较基因组学方法,参考已发表的拟南芥和水稻的磷响应基因,在NCBI数据库中搜索,筛选得到10个与已知磷响应基因同源性较高的小麦ESTs,利用半定量RT-PCR对各候选基因的表达情况进行了分析。结果表明:在候选基因中有4个基因显示出磷胁迫下转录增强,显示其可能在磷调控基因网络中发挥重要作用;其中,CJ916459和CJ895163基因的转录水平在小麦根系中显著高于地上部,而DQ512334和BE418377基因则相反,但BE418377基因表达量均相对较弱。研究结果可为进一步分析磷代谢相关基因表达网络和克隆磷高效关键基因提供参考。
A wheat hydroponic culture system was established for detecting the wheat phosphate stress and screening for the response genes of wheat phosphate stress. The candidate house-keeping genes of wheat were identified by measuring the mRNA transcription. The technical system established here would allow the researches of wheat phosphate stress at the nutrition strictly controlling condition. The reached results would profit the discovery of house-keeping genes and phosphate stress response genes of wheat. The technical system and results are important for us to find phosphate high-efficient utilizing genes and screen germplasm resource of wheat. The main results as follows:
1、Modified Hoagland medium was employed to establish the wheat hydroponic culture system under green house condition. Phosphate stress responses of different genotype wheat were measured by using this system.
(1)Under the hydroponic culture established in this research, the experimental wheat plants grew well and performed the typical architecture responses when exposed in Pi deprivation. Hemi-quantitative RT-PCR revealed that the transcriptional level of known Pi response gene differed dramatically.
(2)In general the root length of wheat and root/shoot ratio(R/S) arose when the Pi supply was deprived, however, the change models differed among different wheat genotypes: In tested varieties, Chinese spring and Zhou-mai 18 respond to phosphate stress more slowly, Tai-kong 6hao、Yu-nong 202、Yan-zhan 4110 made early response of phosphate stress, following to display trend of platform; A valuable point is that Zheng-mai 9023 showed a manifestly recovery after a morphological response to the Pi deficiency, it might indicate that there existed possible mechanism to reimburse the lasting Pi starvation. The investigation on their molecular basis would offer the useful clue for screening phosphate high-efficient gene resources of wheat and analyzing gene net controlling metabolism of wheat.
2、According to the published house-keeping genes of Arabidopsis, ten genes of wheat with high sequence similarity to published genes were obtained by searching in NCBI database. We analyzed expression of candidate genes in space time of different genotypes wheat by Hemi-quantitative RT-PCR. The results showed that ubiquitin-conjugating enzyme(AY736121)、translation elongation factor 1 alpha-subunit(M90077)、delta-type tonoplast intrinsic protein (U86763) expressed steadily in different organs of Yanzhan 4110 and Zhengmai 9023 in different periods. Three genes have the potential applicability.
3、Using comparative genomics method, according to the published phosphate response genes of Arabidopsis and Rice, ten genes of wheat with high sequence similarity to published genes were obtained by searching in NCBI database. We analyzed expression of candidate genes by Hemi-quantitative RT-PCR. The results showed that the four genes of candidate had increased transcription in response to phosphate stress, they were suggested to be important to gene net regulated by phosphate. Further more, transcription of CJ916459 and CJ895163 increased more in root than in shoot,while DQ512334 and BE418377 were opposite, and BE418377 had a low transcription generally. The results gave us references to farther analyze gene expressing net controlling metabolism of phosphate and clone phosphate high-efficient utilizing genes.
1、采用改进的Hoagland培养基,建立并优化了小麦温室水培体系,对不同基因型小麦在水培条件下对磷饥饿胁迫的生理反应进行了研究。
(1)在建立的水培体系中,小麦植株生长正常并对磷饥饿胁迫条件做出形态建成水平的反应;半定量RT-PCR分析显示在此体系中小麦磷响应基因在转录水平上差异显著。
(2)在水培条件下,不同基因型小麦对磷饥饿胁迫的反应总体上表现在根系长度和根冠比所提高,但不同基因型小麦的反应特点有差异即在供试品种中,中国春和周麦18对磷饥饿做出的反应较为迟缓,而太空6号、豫农202和偃展4110则在较早时间对磷饥饿做出发育水平反应,随后呈现平台趋势;值得注意的是郑麦9023在对磷饥饿较早做出形态适应反应后出现明显恢复,暗示其可能存在某种显著的补偿机制。对这些反应特征的分子机理的深入分析,可为深入解析小麦代谢调控的基因网络和有效筛选磷高效小麦基因资源提供线索。
2、参考已经发表的部分拟南芥看家基因,在NCBI数据库中搜索得到10个小麦同源性较高的ESTs,通过半定量RT-PCR方法,分析了各候选基因在不同基因型小麦(偃展4110、郑麦9023)中的时空表达情况。结果表明:小麦泛肽结合酶基因(AY736121)、翻译延伸因子I-α亚基基因(M90077)和δ-液泡膜内蛋白基因(U86763)在偃展4110和郑麦9023小麦各个时期不同组织中均稳定表达,具有作为看家基因的应用潜力。
3、利用比较基因组学方法,参考已发表的拟南芥和水稻的磷响应基因,在NCBI数据库中搜索,筛选得到10个与已知磷响应基因同源性较高的小麦ESTs,利用半定量RT-PCR对各候选基因的表达情况进行了分析。结果表明:在候选基因中有4个基因显示出磷胁迫下转录增强,显示其可能在磷调控基因网络中发挥重要作用;其中,CJ916459和CJ895163基因的转录水平在小麦根系中显著高于地上部,而DQ512334和BE418377基因则相反,但BE418377基因表达量均相对较弱。研究结果可为进一步分析磷代谢相关基因表达网络和克隆磷高效关键基因提供参考。
A wheat hydroponic culture system was established for detecting the wheat phosphate stress and screening for the response genes of wheat phosphate stress. The candidate house-keeping genes of wheat were identified by measuring the mRNA transcription. The technical system established here would allow the researches of wheat phosphate stress at the nutrition strictly controlling condition. The reached results would profit the discovery of house-keeping genes and phosphate stress response genes of wheat. The technical system and results are important for us to find phosphate high-efficient utilizing genes and screen germplasm resource of wheat. The main results as follows:
1、Modified Hoagland medium was employed to establish the wheat hydroponic culture system under green house condition. Phosphate stress responses of different genotype wheat were measured by using this system.
(1)Under the hydroponic culture established in this research, the experimental wheat plants grew well and performed the typical architecture responses when exposed in Pi deprivation. Hemi-quantitative RT-PCR revealed that the transcriptional level of known Pi response gene differed dramatically.
(2)In general the root length of wheat and root/shoot ratio(R/S) arose when the Pi supply was deprived, however, the change models differed among different wheat genotypes: In tested varieties, Chinese spring and Zhou-mai 18 respond to phosphate stress more slowly, Tai-kong 6hao、Yu-nong 202、Yan-zhan 4110 made early response of phosphate stress, following to display trend of platform; A valuable point is that Zheng-mai 9023 showed a manifestly recovery after a morphological response to the Pi deficiency, it might indicate that there existed possible mechanism to reimburse the lasting Pi starvation. The investigation on their molecular basis would offer the useful clue for screening phosphate high-efficient gene resources of wheat and analyzing gene net controlling metabolism of wheat.
2、According to the published house-keeping genes of Arabidopsis, ten genes of wheat with high sequence similarity to published genes were obtained by searching in NCBI database. We analyzed expression of candidate genes in space time of different genotypes wheat by Hemi-quantitative RT-PCR. The results showed that ubiquitin-conjugating enzyme(AY736121)、translation elongation factor 1 alpha-subunit(M90077)、delta-type tonoplast intrinsic protein (U86763) expressed steadily in different organs of Yanzhan 4110 and Zhengmai 9023 in different periods. Three genes have the potential applicability.
3、Using comparative genomics method, according to the published phosphate response genes of Arabidopsis and Rice, ten genes of wheat with high sequence similarity to published genes were obtained by searching in NCBI database. We analyzed expression of candidate genes by Hemi-quantitative RT-PCR. The results showed that the four genes of candidate had increased transcription in response to phosphate stress, they were suggested to be important to gene net regulated by phosphate. Further more, transcription of CJ916459 and CJ895163 increased more in root than in shoot,while DQ512334 and BE418377 were opposite, and BE418377 had a low transcription generally. The results gave us references to farther analyze gene expressing net controlling metabolism of phosphate and clone phosphate high-efficient utilizing genes.
引文
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