ICE1-like转录因子的克隆及其转化水稻的抗冷效果评价
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摘要
针对寒地水稻冷害频发的特点及转录因子在植物对低温的反应中起着重要的调控作用，本研究：(1)从三种抗寒植物中分离了bHLH转录因子家族中与冷胁迫相关的三个ICE1-like基因，并进行了序列分析和功能预测。(2)分析了ICE1-like基因的组织及相应胁迫表达模式，对基因进行了原核表达，并利用酵母单杂交对其转录激活域进行了验证。(3)构建植物表达载体对水稻进行遗传转化，利用超表达ICE1-like基因的转基因水稻株系对所克隆基因进行了功能验证，对其在转基因水稻中的抗冷效果进行评价及冷反应通路的调控模式进行分析，以期获得在水稻冷害改良方面具有应用潜力的候选基因，为培育抗冷害水稻品种奠定理论基础。主要研究结果如下：
(1)利用SON-PCR和RT-PCR技术从生菜中分离到一个ICE1-like基因，命名为LsICE1（获得基因登录号为HQ848932），其cDNA片段长1622bp，含有一个1497bp完整的开放阅读框，编码498个氨基酸；利用SON-PCR和RT-PCR技术从大白菜中分离到一个ICE1-like基因，命名为BcICE1（获得基因登录号为HQ902162），其cDNA片段长1591bp，含有一个1494bp完整的开放阅读框，编码498个氨基酸；利用电子克隆和RT-PCR技术从萝卜中分离到一个ICE1-like基因，命名为RsICE1（获得基因登录号为HQ891287），其cDNA片段长1375bp，含有一个1266bp完整的开放阅读框，编码421个氨基酸。LsICE1、BcICE1、RsICE1蛋白与拟南芥ICE1蛋白一级结构和二级结构极其相似，与其它植物ICE1-like家族蛋白的C末端区域一致性很高，并且具有高度保守的bHLH结构域、SUMO结合位点和核定位信号区。其中，LsICE1蛋白与葡萄的ICE1-like蛋白处于同一进化分枝；BcICE1蛋白与山嵛菜及盐芥菜的ICE1-like蛋白处于同一进化分枝；RsICE1蛋白与山嵛菜等十字花科的ICE1-like蛋白处于同一进化分枝。
(2)组织表达分析表明，LsICE1、BcICE1、RsICE1基因在生菜、大白菜和萝卜的根、茎、叶中均有表达。其中，LsICE1基因在生菜幼苗叶的表达量最强，BcICE1基因在大白菜幼苗茎的表达量最强，而RsICE1基因在萝卜幼苗根的表达量最强。诱导表达分析表明，在低温（4℃）和高盐（200mM NaCl）胁迫处理的幼苗中，LsICE1、BcICE1、RsICE1基因表达量呈现是先上调后下降趋势，说明低温和高盐处理都能影响基因的表达。100μMABA胁迫处理能上调LsICE1、BcICE1基因表达量，但对RsICE1基因表达量影响较小。此外，7%PEG6000干旱脱水处理对LsICE1、BcICE1、RsICE1基因的表达量影响不大，说明三个基因对脱水胁迫不敏感。
(3)构建了LsICE1、 BcICE1、 RsICE1基因原核表达载体pET32a-LsICE1、pET32a-BcICE1、pET32a-RsICE1，转化大肠杆菌BL21（DE3），筛选重组菌株，经IPTG诱导后，SDS-PAGE检测到大小与预测一致的特异蛋白产物。构建携带LsICE1、BcICE1、RsICE1基因转录激活载体进行酵母单杂交，酵母转化菌株可以在SD/-Trp平板上生长，并能被X-Gal染成蓝色，表明LsICE1、BcICE1、RsICE1基因可以激活酵母下游的报告基因表达，具有转录激活功能。
(4)构建了LsICE1、BcICE1、RsICE1基因超表达载体，通过农杆菌介导法，将LsICE1、BcICE1、RsICE1基因导入单子叶模式植物水稻中，使LsICE1、BcICE1、RsICE1基因在水稻中超量表达。通过PCR、PCR-Southern和RT-PCR检测，共获得47株外源基因超表达的转基因水稻植株。其中，14株转LsICE1基因水稻植株，16株转BcICE1基因水稻植株，17株转RsICE1基因水稻植株。T_0代转基因水稻的生长速度比非转基因水稻慢，生育期延迟，转基因植株的株高、穗长、穗粒数、千粒重和结实率与非转基因植株有较大差异。
(5) χ2测验结果表明，33个T_0代转基因植株自交种子（T_1代）均发生了潮霉素抗性分离。其中，13个植株种子的潮霉素抗感分离比均不符合3:1，目的基因可能是以非单拷贝单位点形式整合，20个植株种子潮霉素抗感分离比符合3:1，目的基因可能是以单拷贝单位点形式整合。
(6)低温恢复生长法测定转基因株系在冷胁迫下的存活率，有11个株系较未转基因对照表现出明显的抗寒性（P<0.01）。11个转基因水稻株系存活率大小依次为：T_1L11>T_1L7>T_1L26>T_1L2>T_1L37>T_1L21>T_1L32>T_1L27> T_1L15>T_1L30>T_1L19。其中，转LsICE1基因抗寒水稻株系存活率>转BcICE1基因抗寒水稻株系存活率>转RsICE1基因抗寒水稻株系存活率。
(7)在低温胁迫过程中，转基因株系的相对电导率和丙二醛含量积累速率明显低于非转基因对照。低温胁迫72h后，11个转基因抗寒水稻株系最终相对电导率大小依次为：T_1L19>T_1L27>T_1L30>T_1L32>T_1L15>T_1L2>T_1L21>T_1L26>T_1L7>T_1L11>T_1L37，均显著低于未转基因对照（P<0.01）。11个转基因抗寒水稻株系最终相对电导率增幅大小依次为：T_1L19>T_1L30>T_1L27>T_1L15>T_1L32>T_1L37>T_1L21>T_1L2>T_1L7>T_1L26>T_1L11，均低于未转基因对照增幅。其中，转RsICE1基因抗寒水稻株系相对电导率增幅>转BcICE1基因抗寒水稻株系相对电导率增幅>转LsICE1基因抗寒水稻株系相对电导率增幅。11个转基因抗寒水稻株系最终丙二醛含量大小依次为：T_1L19>T_1L30>T_1L27>T_1L15>T_1L32>T_1L21>T_1L37>T_1L2>T_1L7>T_1L11>T_1L26，均显著低于未转基因对照（P<0.01）。11个转基因抗寒水稻株系最终丙二醛增幅大小依次为：T_1L19>T_1L30>T_1L27>T_1L15>T_1L32>T_1L37>T_1L21>T_1L2>T_1L26>T_1L7>T_1L11，均低于未转基因对照增幅。其中，转RsICE1基因抗寒水稻株系丙二醛增幅>转BcICE1基因抗寒水稻株系丙二醛增幅>转LsICE1基因抗寒水稻株系丙二醛增幅。
(8)低温胁迫下，LsICE1、BcICE1、RsICE1基因能促进转基因水稻脯氨酸大量合成。转基因抗寒水稻株系最终游离脯氨酸含量大小依次为：T_1L27>T_1L21>T_1L15>T_1L7>T_1L2>T_1L11>T_1L26>T_1L32>T_1L37>T_1L19>T_1L30，显著高于未转基因对照（P<0.01）。转基因抗寒水稻株系最终游离脯氨酸增幅大小依次为：T_1L27>T_1L2>T_1L15>T_1L21>T_1L7>T_1L11>T_1L26>T_1L30>T_1L32>T_1L37>T_1L19，高于未转基因对照增幅。其中，转RsICE1基因抗寒水稻株系脯氨酸增幅>转LsICE1基因抗寒水稻株系脯氨酸增幅>转BcICE1基因抗寒水稻株系脯氨酸增幅。
(9) Southern blot分析结果显示，转LsICE1基因抗寒水稻株系（T_1L2、T_1L7和T_1L11）、转BcICE1基因抗寒水稻株系（T_1L15、T_1L19、T_1L21和T_1L26）和转RsICE1基因抗寒水稻株系（T_1L27、T_1L30、T_1L32和T_1L37）中的目的基因均是以单拷贝单位点形式整合，与χ2测验结果完全一致。Northern blot进一步分析结果显示，目的基因LsICE1、BcICE1、RsICE1基因在11个T_1代转基因抗寒水稻株系中能够正常表达。
(10)利用实时定量PCR对转基因水稻冷胁迫转录因子OsDREB1F、OsDREB1B、OsDREB1A基因进行表达分析表明，在冷胁迫下，LsICE1、BcICE1基因对转基因水稻抗寒性的影响是依赖CBF/DREB1冷反应通路的，而RsICE1基因对转基因水稻抗寒性的影响是不依赖CBF/DREB1冷反应通路的。其中，LsICE1基因能够显著上调OsDREB1A基因的转录水平（P<0.05），而BcICE1基因能够显著上调OsDREB1F基因的转录水平（P<0.05）。
In view of chilling damages happening frequently for rice and the important regulationof transcription factors in plant responses to low temperature, the following studies hawe beencarried out in this study:(1) Three ICE1-like genes related cold stress, members of bHLHfamily genes, were isolated from three cold-resistance plants respectively, the proteinsequences encoded by genes were analyzed, and the protein functions were predicted.(2) Themodes of tissue expression and stress expression for ICE1-like genes were analyzed,prokaryotic expression of genes was conducted, and yeast-one-hybrid system was used tovalidate transactivation function regions of ICE1-like genes.(3) Constructing plant expressionvectors and transformation into rice, the transgenic rice lines with overexpression ofICE1-like genes were used to validate the function of the genes, and the cold-resistanceeffects and regulation modes of cold responsive pathways for the genes in transgenic ricewere evaluated and analyzed respectively, expecting to provide new candidate genes forimproving rice in cold resistance and the theoretical basis for breeding rice varieties with coldresistance. The main results are as follows:
(1) One ICE1-like gene, designated as LsICE1(GenBank accession No. HQ848932), wasisolated from lettuce by single oligonucleotide nested PCR (SON-PCR) and reversetranscription-PCR (RT-PCR). Sequence analysis showed that the LsICE1cDNA fragmentlength was1622bp containing a full coding region of1497bp encoding498amino acidresidues. The other ICE1-like gene, designated as BcICE1(GenBank accession No.HQ902162), was isolated from Chinese cabbage by SON-PCR and RT-PCR. The full-lengthcDNA of BcICE1was1591bp with a1494bp open reading frame (ORF) encoding497aminoacid residues. Another ICE1-like gene, designated as RsICE1(GenBank accession No.HQ891287), was isolated from radish by in silicon cloning and RT-PCR. Sequence analysisshowed that the full-length cDNA of RsICE1was1375bp and contained a complete codingregion of1266bp, which encoded421amino acid residues. Sequence multialigmentdemonstrated that the primary structures and secondary structure of the LsICE1, BcICE1,RsICE1proteins were extremly similar to that of Arabidopsis thaliana. The C-terminal of deduced LsICE1, BcICE1, RsICE1proteins were high identity with other ICE1-like proteinsfrom different species and exhibited a typical bHLH domain, SUMO binding site and nuclearlocalization signal (NLS). The homology tree showed that LsICE1protein was at the sameevolutionary branch with ICE1-like protein of Vitis vinifera, BcICE1protein was at the sameevolutionary branch with ICE1-like proteins of Eutrema salsugineum and Thellungiellahalophila, and RsICE1protein was at the same evolutionary branch with ICE1-like proteins ofCruciferae plants, such as Eutrema salsugineum.
(2) The results showed that LsICE1, BcICE1and RsICE1genes were expressedconstitutively in the examined tissues, such as the leaves, roots and stems of lettuce, Chinesecabbage and radish. LsICE1mRNA levels were dominant in leaves of lettuce seedling,BcICE1mRNA levels were dominant in stems of Chinese Cabbage seedling, while RsICE1gene mRNA levels were dominant in roots of radish seedling. Analysis of inductionexpression indicated that the mRNA levels of LsICE1, BcICE1, RsICE1genes were firstup-regulated then down-regulated in the seedlings under the low temperature stress (4°C) andhigh salt (200mM NaCl) treatments, which proved that both low-temperature and high salteffected the expression of genes. Under100μM ABA stress, the expression of LsICE1andBcICE1was up-regulated, but such stress had little effect on the expression of RsICE1gene.In addition, by time of exposure to the7%PEG6000dehydration stress, the expression ofLsICE1, BcICE1, RsICE1genes had little change observed, suggesting that the expression ofLsICE1, BcICE1, RsICE1genes was insensitive to dehydration stress.
(3) The prokaryotic expression vectors for LsICE1, BcICE1, RsICE1genes respectively,pET32a-LsICE1, pET32a-BcICE1, pET32a-RsICE1, were constructed, then they weretransferred into E.coli BL21(DE3), recombined strains were screened, and special proteinproduct with the same molecular weight as prediction was detected by SDS-PAGE afterinduced by IPTG. Transcription activation expression vectors with LsICE1, BcICE1, RsICE1gene respectively were constructed to carry out yeast-one-hybridization. The recombinantyeast strains could grow on SD/-Trp plate, and could be dyed blue by X-Gal, suggesting thatLsICE1, BcICE1, RsICE1genes could activate the expression of downstream reporter gene ofyeast, and owned the function of transcriptional activation.
(4) The over-expression vectors of LsICE1, BcICE1, RsICE1genes were constructedrespectively, then LsICE1, BcICE1, RsICE1genes were transferred into rice asmonocotyledonous model plant by Agrobacterium mediated genetic transformation system,leading to over-expression of LsICE1, BcICE1, RsICE1genes in rice. A total of47transgenicrice plants with over-expression exogenous genes were obtained by the PCR, PCR-Southernand RT-PCR. detection. Among these plants, there were14transgenic rice plants with LsICE1gene,16transgenic rice plants with BcICE1gene,17transgenic rice plants with RsICE1gene,respectively. T_0transgenic rice plants grew slower, and had a delay of growth anddevelopment period than non-transgenic rice plants. The plant height, panicle length, numberof grains per panicle, seed setting rate and1000-grain weight of transgenic rice plants hadsignificant differences from those of non-transgenic plants.
(5) χ~2test indicated that the Hyg-resistance segregation occurred in self-pollinated seeds(T_1generation) of33T_0rice plants. Among them, the Hyg-resistance segregations of13transgenic rice lines were not in accord with3:1which showed that target genes weretransformed to rice by not a single copy and a single site, while the Hyg-resistancesegregations of20rice lines were3:1, suggesting that target genes were transformed to riceby a single copy and a single site.
(6) Low temperature recovering growth method was used to test the survival rates oftransgenic rice lines under cold stress,11transgenic rice lines showed obvious cold resistancethan non-transgenic rice lines (P<0.01). The survival rates of them were as follows:T_1L11>T_1L7>T_1L26>T_1L2>T_1L37>T_1L21>T_1L32>T_1L27>T_1L15>T_1L30>T_1L19. In addition,the transgenic rice lines with LsICE1gene had higher survival rates than the transgenic ricelines with BcICE1gene, while the transgenic rice lines with BcICE1gene had higher survivalrates than transgenic rice lines with RsICE1gene.
(7) Under low temperature stress treatment, compared to control, the transgenic lines hadlower accumulation rate of relative conductivity and malondialdehyde (MDA) content. Afterlow temperature stress for72h, the final relative electric conductivity of11transgenic ricelines with cold resistance was as follows: T_1L19>T_1L27>T_1L30>T_1L32>T_1L15>T_1L2>T_1L21>T_1L26>T_1L7>T_1L11>T_1L37, which was significantly lower than that of the non-transgenic control (P<0.01). The final relative electric conductivity increase amplitude of11transgenicrice lines with cold resistance was as follows: T_1L19>T_1L30>T_1L27>T_1L15>T_1L32>T_1L37>T_1L21>T_1L2>T_1L7>T_1L26>T_1L11, which was significantly lower than that of thenon-transgenic control. The transgenic rice lines with RsICE1gene had higher relative electricconductivity increase amplitude than the transgenic rice lines with BcICE1gene, while thetransgenic rice lines with BcICE1gene had higher relative electric conductivity increaseamplitude than transgenic rice lines with LsICE1gene.The final malonyldialdehyde (MDA)content of11transgenic plants with cold resistance was as follows: T_1L19>T_1L30>T_1L27>T_1L15>T_1L32>T_1L21>T_1L37>T_1L2>T_1L7>T_1L11>T_1L26, which was significantly lowerthan that of the non-transgenic control (P<0.01). The final MDA increase amplitude of11transgenic rice lines with cold resistance was as follows: T_1L19>T_1L30>T_1L27>T_1L15>T_1L32>T_1L37>T_1L21>T_1L2>T_1L26>T_1L7>T_1L11, which was significantly lower than that ofthe non-transgenic control. The transgenic rice lines with RsICE1gene had higher MDAincrease amplitude than the transgenic rice lines with BcICE1gene, while the transgenic ricelines with BcICE1gene had higher MDA increase amplitude than transgenic rice lines withLsICE1gene.
(8) Under low temperature stress, LsICE1, BcICE1, RsICE1gene promoted the masssynthesis of proline in transgenic plants. The final free proline content of11transgenic ricelines with cold resistance was as follows: T_1L27>T_1L21>T_1L15>T_1L7>T_1L2>T_1L11>T_1L26>T_1L32>T_1L37>T_1L19>T_1L30, which was significantly higher than that of the non-transgeniccontrol (P<0.01). The final free proline content increase amplitude of11transgenic rice lineswith cold resistance was as follow: T_1L27>T_1L2>T_1L15>T_1L21>T_1L7>T_1L11>T_1L26>T_1L30>T_1L32>T_1L37>T_1L19>T_1L19, which was significantly higher than that of thenon-transgenic control. The transgenic rice lines with RsICE1gene had higher prolineincrease amplitude than the transgenic rice lines with LsICE1gene, while the transgenic ricelines with LsICE1gene had higher MDA increase amplitude than transgenic rice lines withBcICE1gene.
(9) Southern blot analysis showed that the target genes of transgenic rice lines withLsICE1gene (T_1L2, T_1L7, T_1L11), transgenic rice lines with BcICE1gene (T_1L15, T_1L19, T_1L21, T_1L26) and transgenic rice lines with RsICE1gene (T_1L27, T_1L30, T_1L32, T_1L37)were transformed to rice by a single copy and a single site, which was completely in accordwith the results of χ2test. Northern blot analysis further indicated that LsICE1, BcICE1,RsICE1genes were able to express normally in11T_1rice lines.
(10) Real time quantitative RT-PCR was used to analyze the expression levels of coldstress-inducible transcription factors, OsDREB1F, OsDREB1B and OsDREB1A genes, intransgenic rice lines. The expression analysis of OsDREB1F, OsDREB1B and OsDREB1Agenes showed that the effects of LsICE1, BcICE1genes on the cold resistance of transgenicrice lines were dependent on CBF/DREB1cold responsive pathway, but the effect ofRsICE1genes on the cold resistance of transgenic rice lines was independent onCBF/DREB1cold responsive pathway under the cold stress. In addition, LsICE1gene wasable to upregulate significantly the transcriptional levels of OsDREB1A gene (P<0.05), whileBcICE1gene was able to upregulate significantly the transcriptional levels of OsDREB1Fgene (P<0.05).
(1)利用SON-PCR和RT-PCR技术从生菜中分离到一个ICE1-like基因，命名为LsICE1（获得基因登录号为HQ848932），其cDNA片段长1622bp，含有一个1497bp完整的开放阅读框，编码498个氨基酸；利用SON-PCR和RT-PCR技术从大白菜中分离到一个ICE1-like基因，命名为BcICE1（获得基因登录号为HQ902162），其cDNA片段长1591bp，含有一个1494bp完整的开放阅读框，编码498个氨基酸；利用电子克隆和RT-PCR技术从萝卜中分离到一个ICE1-like基因，命名为RsICE1（获得基因登录号为HQ891287），其cDNA片段长1375bp，含有一个1266bp完整的开放阅读框，编码421个氨基酸。LsICE1、BcICE1、RsICE1蛋白与拟南芥ICE1蛋白一级结构和二级结构极其相似，与其它植物ICE1-like家族蛋白的C末端区域一致性很高，并且具有高度保守的bHLH结构域、SUMO结合位点和核定位信号区。其中，LsICE1蛋白与葡萄的ICE1-like蛋白处于同一进化分枝；BcICE1蛋白与山嵛菜及盐芥菜的ICE1-like蛋白处于同一进化分枝；RsICE1蛋白与山嵛菜等十字花科的ICE1-like蛋白处于同一进化分枝。
(2)组织表达分析表明，LsICE1、BcICE1、RsICE1基因在生菜、大白菜和萝卜的根、茎、叶中均有表达。其中，LsICE1基因在生菜幼苗叶的表达量最强，BcICE1基因在大白菜幼苗茎的表达量最强，而RsICE1基因在萝卜幼苗根的表达量最强。诱导表达分析表明，在低温（4℃）和高盐（200mM NaCl）胁迫处理的幼苗中，LsICE1、BcICE1、RsICE1基因表达量呈现是先上调后下降趋势，说明低温和高盐处理都能影响基因的表达。100μMABA胁迫处理能上调LsICE1、BcICE1基因表达量，但对RsICE1基因表达量影响较小。此外，7%PEG6000干旱脱水处理对LsICE1、BcICE1、RsICE1基因的表达量影响不大，说明三个基因对脱水胁迫不敏感。
(3)构建了LsICE1、 BcICE1、 RsICE1基因原核表达载体pET32a-LsICE1、pET32a-BcICE1、pET32a-RsICE1，转化大肠杆菌BL21（DE3），筛选重组菌株，经IPTG诱导后，SDS-PAGE检测到大小与预测一致的特异蛋白产物。构建携带LsICE1、BcICE1、RsICE1基因转录激活载体进行酵母单杂交，酵母转化菌株可以在SD/-Trp平板上生长，并能被X-Gal染成蓝色，表明LsICE1、BcICE1、RsICE1基因可以激活酵母下游的报告基因表达，具有转录激活功能。
(4)构建了LsICE1、BcICE1、RsICE1基因超表达载体，通过农杆菌介导法，将LsICE1、BcICE1、RsICE1基因导入单子叶模式植物水稻中，使LsICE1、BcICE1、RsICE1基因在水稻中超量表达。通过PCR、PCR-Southern和RT-PCR检测，共获得47株外源基因超表达的转基因水稻植株。其中，14株转LsICE1基因水稻植株，16株转BcICE1基因水稻植株，17株转RsICE1基因水稻植株。T_0代转基因水稻的生长速度比非转基因水稻慢，生育期延迟，转基因植株的株高、穗长、穗粒数、千粒重和结实率与非转基因植株有较大差异。
(5) χ2测验结果表明，33个T_0代转基因植株自交种子（T_1代）均发生了潮霉素抗性分离。其中，13个植株种子的潮霉素抗感分离比均不符合3:1，目的基因可能是以非单拷贝单位点形式整合，20个植株种子潮霉素抗感分离比符合3:1，目的基因可能是以单拷贝单位点形式整合。
(6)低温恢复生长法测定转基因株系在冷胁迫下的存活率，有11个株系较未转基因对照表现出明显的抗寒性（P<0.01）。11个转基因水稻株系存活率大小依次为：T_1L11>T_1L7>T_1L26>T_1L2>T_1L37>T_1L21>T_1L32>T_1L27> T_1L15>T_1L30>T_1L19。其中，转LsICE1基因抗寒水稻株系存活率>转BcICE1基因抗寒水稻株系存活率>转RsICE1基因抗寒水稻株系存活率。
(7)在低温胁迫过程中，转基因株系的相对电导率和丙二醛含量积累速率明显低于非转基因对照。低温胁迫72h后，11个转基因抗寒水稻株系最终相对电导率大小依次为：T_1L19>T_1L27>T_1L30>T_1L32>T_1L15>T_1L2>T_1L21>T_1L26>T_1L7>T_1L11>T_1L37，均显著低于未转基因对照（P<0.01）。11个转基因抗寒水稻株系最终相对电导率增幅大小依次为：T_1L19>T_1L30>T_1L27>T_1L15>T_1L32>T_1L37>T_1L21>T_1L2>T_1L7>T_1L26>T_1L11，均低于未转基因对照增幅。其中，转RsICE1基因抗寒水稻株系相对电导率增幅>转BcICE1基因抗寒水稻株系相对电导率增幅>转LsICE1基因抗寒水稻株系相对电导率增幅。11个转基因抗寒水稻株系最终丙二醛含量大小依次为：T_1L19>T_1L30>T_1L27>T_1L15>T_1L32>T_1L21>T_1L37>T_1L2>T_1L7>T_1L11>T_1L26，均显著低于未转基因对照（P<0.01）。11个转基因抗寒水稻株系最终丙二醛增幅大小依次为：T_1L19>T_1L30>T_1L27>T_1L15>T_1L32>T_1L37>T_1L21>T_1L2>T_1L26>T_1L7>T_1L11，均低于未转基因对照增幅。其中，转RsICE1基因抗寒水稻株系丙二醛增幅>转BcICE1基因抗寒水稻株系丙二醛增幅>转LsICE1基因抗寒水稻株系丙二醛增幅。
(8)低温胁迫下，LsICE1、BcICE1、RsICE1基因能促进转基因水稻脯氨酸大量合成。转基因抗寒水稻株系最终游离脯氨酸含量大小依次为：T_1L27>T_1L21>T_1L15>T_1L7>T_1L2>T_1L11>T_1L26>T_1L32>T_1L37>T_1L19>T_1L30，显著高于未转基因对照（P<0.01）。转基因抗寒水稻株系最终游离脯氨酸增幅大小依次为：T_1L27>T_1L2>T_1L15>T_1L21>T_1L7>T_1L11>T_1L26>T_1L30>T_1L32>T_1L37>T_1L19，高于未转基因对照增幅。其中，转RsICE1基因抗寒水稻株系脯氨酸增幅>转LsICE1基因抗寒水稻株系脯氨酸增幅>转BcICE1基因抗寒水稻株系脯氨酸增幅。
(9) Southern blot分析结果显示，转LsICE1基因抗寒水稻株系（T_1L2、T_1L7和T_1L11）、转BcICE1基因抗寒水稻株系（T_1L15、T_1L19、T_1L21和T_1L26）和转RsICE1基因抗寒水稻株系（T_1L27、T_1L30、T_1L32和T_1L37）中的目的基因均是以单拷贝单位点形式整合，与χ2测验结果完全一致。Northern blot进一步分析结果显示，目的基因LsICE1、BcICE1、RsICE1基因在11个T_1代转基因抗寒水稻株系中能够正常表达。
(10)利用实时定量PCR对转基因水稻冷胁迫转录因子OsDREB1F、OsDREB1B、OsDREB1A基因进行表达分析表明，在冷胁迫下，LsICE1、BcICE1基因对转基因水稻抗寒性的影响是依赖CBF/DREB1冷反应通路的，而RsICE1基因对转基因水稻抗寒性的影响是不依赖CBF/DREB1冷反应通路的。其中，LsICE1基因能够显著上调OsDREB1A基因的转录水平（P<0.05），而BcICE1基因能够显著上调OsDREB1F基因的转录水平（P<0.05）。
In view of chilling damages happening frequently for rice and the important regulationof transcription factors in plant responses to low temperature, the following studies hawe beencarried out in this study:(1) Three ICE1-like genes related cold stress, members of bHLHfamily genes, were isolated from three cold-resistance plants respectively, the proteinsequences encoded by genes were analyzed, and the protein functions were predicted.(2) Themodes of tissue expression and stress expression for ICE1-like genes were analyzed,prokaryotic expression of genes was conducted, and yeast-one-hybrid system was used tovalidate transactivation function regions of ICE1-like genes.(3) Constructing plant expressionvectors and transformation into rice, the transgenic rice lines with overexpression ofICE1-like genes were used to validate the function of the genes, and the cold-resistanceeffects and regulation modes of cold responsive pathways for the genes in transgenic ricewere evaluated and analyzed respectively, expecting to provide new candidate genes forimproving rice in cold resistance and the theoretical basis for breeding rice varieties with coldresistance. The main results are as follows:
(1) One ICE1-like gene, designated as LsICE1(GenBank accession No. HQ848932), wasisolated from lettuce by single oligonucleotide nested PCR (SON-PCR) and reversetranscription-PCR (RT-PCR). Sequence analysis showed that the LsICE1cDNA fragmentlength was1622bp containing a full coding region of1497bp encoding498amino acidresidues. The other ICE1-like gene, designated as BcICE1(GenBank accession No.HQ902162), was isolated from Chinese cabbage by SON-PCR and RT-PCR. The full-lengthcDNA of BcICE1was1591bp with a1494bp open reading frame (ORF) encoding497aminoacid residues. Another ICE1-like gene, designated as RsICE1(GenBank accession No.HQ891287), was isolated from radish by in silicon cloning and RT-PCR. Sequence analysisshowed that the full-length cDNA of RsICE1was1375bp and contained a complete codingregion of1266bp, which encoded421amino acid residues. Sequence multialigmentdemonstrated that the primary structures and secondary structure of the LsICE1, BcICE1,RsICE1proteins were extremly similar to that of Arabidopsis thaliana. The C-terminal of deduced LsICE1, BcICE1, RsICE1proteins were high identity with other ICE1-like proteinsfrom different species and exhibited a typical bHLH domain, SUMO binding site and nuclearlocalization signal (NLS). The homology tree showed that LsICE1protein was at the sameevolutionary branch with ICE1-like protein of Vitis vinifera, BcICE1protein was at the sameevolutionary branch with ICE1-like proteins of Eutrema salsugineum and Thellungiellahalophila, and RsICE1protein was at the same evolutionary branch with ICE1-like proteins ofCruciferae plants, such as Eutrema salsugineum.
(2) The results showed that LsICE1, BcICE1and RsICE1genes were expressedconstitutively in the examined tissues, such as the leaves, roots and stems of lettuce, Chinesecabbage and radish. LsICE1mRNA levels were dominant in leaves of lettuce seedling,BcICE1mRNA levels were dominant in stems of Chinese Cabbage seedling, while RsICE1gene mRNA levels were dominant in roots of radish seedling. Analysis of inductionexpression indicated that the mRNA levels of LsICE1, BcICE1, RsICE1genes were firstup-regulated then down-regulated in the seedlings under the low temperature stress (4°C) andhigh salt (200mM NaCl) treatments, which proved that both low-temperature and high salteffected the expression of genes. Under100μM ABA stress, the expression of LsICE1andBcICE1was up-regulated, but such stress had little effect on the expression of RsICE1gene.In addition, by time of exposure to the7%PEG6000dehydration stress, the expression ofLsICE1, BcICE1, RsICE1genes had little change observed, suggesting that the expression ofLsICE1, BcICE1, RsICE1genes was insensitive to dehydration stress.
(3) The prokaryotic expression vectors for LsICE1, BcICE1, RsICE1genes respectively,pET32a-LsICE1, pET32a-BcICE1, pET32a-RsICE1, were constructed, then they weretransferred into E.coli BL21(DE3), recombined strains were screened, and special proteinproduct with the same molecular weight as prediction was detected by SDS-PAGE afterinduced by IPTG. Transcription activation expression vectors with LsICE1, BcICE1, RsICE1gene respectively were constructed to carry out yeast-one-hybridization. The recombinantyeast strains could grow on SD/-Trp plate, and could be dyed blue by X-Gal, suggesting thatLsICE1, BcICE1, RsICE1genes could activate the expression of downstream reporter gene ofyeast, and owned the function of transcriptional activation.
(4) The over-expression vectors of LsICE1, BcICE1, RsICE1genes were constructedrespectively, then LsICE1, BcICE1, RsICE1genes were transferred into rice asmonocotyledonous model plant by Agrobacterium mediated genetic transformation system,leading to over-expression of LsICE1, BcICE1, RsICE1genes in rice. A total of47transgenicrice plants with over-expression exogenous genes were obtained by the PCR, PCR-Southernand RT-PCR. detection. Among these plants, there were14transgenic rice plants with LsICE1gene,16transgenic rice plants with BcICE1gene,17transgenic rice plants with RsICE1gene,respectively. T_0transgenic rice plants grew slower, and had a delay of growth anddevelopment period than non-transgenic rice plants. The plant height, panicle length, numberof grains per panicle, seed setting rate and1000-grain weight of transgenic rice plants hadsignificant differences from those of non-transgenic plants.
(5) χ~2test indicated that the Hyg-resistance segregation occurred in self-pollinated seeds(T_1generation) of33T_0rice plants. Among them, the Hyg-resistance segregations of13transgenic rice lines were not in accord with3:1which showed that target genes weretransformed to rice by not a single copy and a single site, while the Hyg-resistancesegregations of20rice lines were3:1, suggesting that target genes were transformed to riceby a single copy and a single site.
(6) Low temperature recovering growth method was used to test the survival rates oftransgenic rice lines under cold stress,11transgenic rice lines showed obvious cold resistancethan non-transgenic rice lines (P<0.01). The survival rates of them were as follows:T_1L11>T_1L7>T_1L26>T_1L2>T_1L37>T_1L21>T_1L32>T_1L27>T_1L15>T_1L30>T_1L19. In addition,the transgenic rice lines with LsICE1gene had higher survival rates than the transgenic ricelines with BcICE1gene, while the transgenic rice lines with BcICE1gene had higher survivalrates than transgenic rice lines with RsICE1gene.
(7) Under low temperature stress treatment, compared to control, the transgenic lines hadlower accumulation rate of relative conductivity and malondialdehyde (MDA) content. Afterlow temperature stress for72h, the final relative electric conductivity of11transgenic ricelines with cold resistance was as follows: T_1L19>T_1L27>T_1L30>T_1L32>T_1L15>T_1L2>T_1L21>T_1L26>T_1L7>T_1L11>T_1L37, which was significantly lower than that of the non-transgenic control (P<0.01). The final relative electric conductivity increase amplitude of11transgenicrice lines with cold resistance was as follows: T_1L19>T_1L30>T_1L27>T_1L15>T_1L32>T_1L37>T_1L21>T_1L2>T_1L7>T_1L26>T_1L11, which was significantly lower than that of thenon-transgenic control. The transgenic rice lines with RsICE1gene had higher relative electricconductivity increase amplitude than the transgenic rice lines with BcICE1gene, while thetransgenic rice lines with BcICE1gene had higher relative electric conductivity increaseamplitude than transgenic rice lines with LsICE1gene.The final malonyldialdehyde (MDA)content of11transgenic plants with cold resistance was as follows: T_1L19>T_1L30>T_1L27>T_1L15>T_1L32>T_1L21>T_1L37>T_1L2>T_1L7>T_1L11>T_1L26, which was significantly lowerthan that of the non-transgenic control (P<0.01). The final MDA increase amplitude of11transgenic rice lines with cold resistance was as follows: T_1L19>T_1L30>T_1L27>T_1L15>T_1L32>T_1L37>T_1L21>T_1L2>T_1L26>T_1L7>T_1L11, which was significantly lower than that ofthe non-transgenic control. The transgenic rice lines with RsICE1gene had higher MDAincrease amplitude than the transgenic rice lines with BcICE1gene, while the transgenic ricelines with BcICE1gene had higher MDA increase amplitude than transgenic rice lines withLsICE1gene.
(8) Under low temperature stress, LsICE1, BcICE1, RsICE1gene promoted the masssynthesis of proline in transgenic plants. The final free proline content of11transgenic ricelines with cold resistance was as follows: T_1L27>T_1L21>T_1L15>T_1L7>T_1L2>T_1L11>T_1L26>T_1L32>T_1L37>T_1L19>T_1L30, which was significantly higher than that of the non-transgeniccontrol (P<0.01). The final free proline content increase amplitude of11transgenic rice lineswith cold resistance was as follow: T_1L27>T_1L2>T_1L15>T_1L21>T_1L7>T_1L11>T_1L26>T_1L30>T_1L32>T_1L37>T_1L19>T_1L19, which was significantly higher than that of thenon-transgenic control. The transgenic rice lines with RsICE1gene had higher prolineincrease amplitude than the transgenic rice lines with LsICE1gene, while the transgenic ricelines with LsICE1gene had higher MDA increase amplitude than transgenic rice lines withBcICE1gene.
(9) Southern blot analysis showed that the target genes of transgenic rice lines withLsICE1gene (T_1L2, T_1L7, T_1L11), transgenic rice lines with BcICE1gene (T_1L15, T_1L19, T_1L21, T_1L26) and transgenic rice lines with RsICE1gene (T_1L27, T_1L30, T_1L32, T_1L37)were transformed to rice by a single copy and a single site, which was completely in accordwith the results of χ2test. Northern blot analysis further indicated that LsICE1, BcICE1,RsICE1genes were able to express normally in11T_1rice lines.
(10) Real time quantitative RT-PCR was used to analyze the expression levels of coldstress-inducible transcription factors, OsDREB1F, OsDREB1B and OsDREB1A genes, intransgenic rice lines. The expression analysis of OsDREB1F, OsDREB1B and OsDREB1Agenes showed that the effects of LsICE1, BcICE1genes on the cold resistance of transgenicrice lines were dependent on CBF/DREB1cold responsive pathway, but the effect ofRsICE1genes on the cold resistance of transgenic rice lines was independent onCBF/DREB1cold responsive pathway under the cold stress. In addition, LsICE1gene wasable to upregulate significantly the transcriptional levels of OsDREB1A gene (P<0.05), whileBcICE1gene was able to upregulate significantly the transcriptional levels of OsDREB1Fgene (P<0.05).
引文
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