紫花苜蓿MsZFN及拟南芥HST基因鉴定
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摘要
本文以紫花苜蓿和拟南芥为研究对象，主要利用基因工程、转基因技术和表型分析，分析MsZFN和HST的功能。主要试验结果如下：
1.通过PCR扩增获得了MsZFN基因gDNA（登陆号：JX131368）全长6829-bp，cDNA全长1667-bp，含有七个外显子，六个内含子,六个内含子长度分别为：820-bp，494-bp，1825-bp，286-bp，641-bp和1093-bp。
2.采用RT-PCR和qRT-PCR的方法，分析MsZFN基因的表达特征。RT-PCR方法显示，MsZFN在所选的组织中（包括根，茎，叶，花）均有表达，但在叶片中表达量最高，花中表达量较低，尤其是花芽中表达量最低；MsZFN基因受黑暗诱导，随着黑暗时间延长，表达量升高。在有20μM ABA，20μM赤霉素等激素诱导条件下，MsZFN基因表达量没有明显差异，在冷处理条件下，MsZFN基因表达量，没有明显的变化。
3.将MsZFN基因编码区插入pCAMBIA1302中，构建双元表达载体35S::MsZFN，并采用农杆菌介导的方法，成功获得拟南芥和烟草转基因植株。结果显示在拟南芥和烟草中过量表达MsZFN基因，能够明显推迟拟南芥和烟草的开花时间。
4.发现一株拟南芥转基因突变体，表型为：白化苗，植株矮小，叶片很小，叶柄较短，没有分枝，腺毛较少，能抽薹，但是不能形成正常的花器官，植株高度败育，但开花时间提前。通过电子透射显微镜（TEM）和电子扫描显微镜（SEM）得知：突变体不能形成正常的叶绿体结构；叶片腺毛分枝由正常的三个，变为了两个；突变体气孔开放程度较大，气孔不能正常关闭。
5.通过TAIL-PCR，发现：该突变体由于T-DNA插入导致的，插入位置在HST基因（At3g11945）的第二个内含子内。将拟南芥HST基因，导入突变体中，能够使突变体性状回复。通过RNAi方法获得HST基因低表达水平的转基因拟南芥植株。
6.通过实时定量PCR和启动子表达分析，得知：该基因在绿色组织中表达量较高，随着叶片的衰老，基因表达量降低。
7.通过ELISA测试，得知：该突变体脱落酸，赤霉素和玉米素水平严重下降，但赤霉素与ABA比值提高；植物生长素（IAA）水平提高。
In this study, we characterized the MsZFN gene from Medicago sativa (alfalfa) and HST gene fromArabidopsis thaliana by gene genetic engineering, transgenic technology and phenotype analysis. Theresults are showed below:
1. A6,829-bp genomic DNA sequence (Access No.: JX131368) was obtained for MsZFN by PCR.Seven exons making up the1,667bp cDNA sequence of MsZFN were interrupted by six introns of820-bp,494-bp,1825-bp,286-bp,641-bp and1093-bp in length when the cDNA and the gDNAsequences were compared.
2. To determine the expression pattern of the MsZFN gene in alfalfa, RT-PCR and qRT-PCR wereperformed. Using RT-PCR, the MsZFN transcript was detected in all alfalfa organs tested, includingroots, stems, leaves, and in florets before and after flower opening. Transcript levels were strongestin leaves compared with stems and roots and was weakest in floral buds and florets. Expressionwas also increased during growth under continuous dark conditions, but not affected by20μMABA,20μM GA3,3%sugar treatments or cold conditions.
3. For transgenic expression experiments, the full-MsZFN coding domain sequence was inserted intothe pCAMBIA1302to generate binary fusion construct35S::MsZFN. To generate transgenicArabidopsis plants expressing MsZFN, Agrobacterium GV3101transformed with plasmid35S::MsZFN was used to infect Arabidopsis and tobacco. The results showed that overexpressionof MsZFN gene delayed flowering time in transgenic plants.
4. We found one Arabidopsis mutant, and the mutant lines are albino and had shorter roots, fewer roothairs, fewer and smaller leaves with shorter petioles, and reduced trichome density. TEM analysisshowed that the mutants had lost the ability to develop into mature chloroplasts. SEM resultsshowed that mutant trichomes were shorter in length and some trichomes had two branches insteadof three. Stomata had bigger openings, suggesting that stomata may close abnormally.
5. The TAIL-PCR and sequencing showed that the mutant phenotypes were caused by a T-DNAinsertion and the insertion was located at the second intron of the Arabidopsis HST gene(At3g11945). When expressed HST gene in mutants, the HST gene could completely rescue themutant phenotype. Transgenic Arabidopsis with low expressed levels of HST gene by RNAitechnology were obtained.
6. To analyze expression pattern of HST gene, qRT-PCR and promoter expression methods wereperformed. The results showed that HST gene was highly expressed in green tissues and the levelsof HST transcript were highest in non-senescent leaves, and the transcript levels graduallydecreased as leaves began to senesce.
7. Enzyme-linked immunosorbent assays (ELISA) were performed to determine concentrations ofdifferent hormones in WT and mutant plants. A substantial decrease in ABA, GA3and ZRconcentrations occurred in mutant plants compared with WT. This disruption of the HST gene also led to a substantial increase (by41.6%) in the IAA content in mutant.
1.通过PCR扩增获得了MsZFN基因gDNA（登陆号：JX131368）全长6829-bp，cDNA全长1667-bp，含有七个外显子，六个内含子,六个内含子长度分别为：820-bp，494-bp，1825-bp，286-bp，641-bp和1093-bp。
2.采用RT-PCR和qRT-PCR的方法，分析MsZFN基因的表达特征。RT-PCR方法显示，MsZFN在所选的组织中（包括根，茎，叶，花）均有表达，但在叶片中表达量最高，花中表达量较低，尤其是花芽中表达量最低；MsZFN基因受黑暗诱导，随着黑暗时间延长，表达量升高。在有20μM ABA，20μM赤霉素等激素诱导条件下，MsZFN基因表达量没有明显差异，在冷处理条件下，MsZFN基因表达量，没有明显的变化。
3.将MsZFN基因编码区插入pCAMBIA1302中，构建双元表达载体35S::MsZFN，并采用农杆菌介导的方法，成功获得拟南芥和烟草转基因植株。结果显示在拟南芥和烟草中过量表达MsZFN基因，能够明显推迟拟南芥和烟草的开花时间。
4.发现一株拟南芥转基因突变体，表型为：白化苗，植株矮小，叶片很小，叶柄较短，没有分枝，腺毛较少，能抽薹，但是不能形成正常的花器官，植株高度败育，但开花时间提前。通过电子透射显微镜（TEM）和电子扫描显微镜（SEM）得知：突变体不能形成正常的叶绿体结构；叶片腺毛分枝由正常的三个，变为了两个；突变体气孔开放程度较大，气孔不能正常关闭。
5.通过TAIL-PCR，发现：该突变体由于T-DNA插入导致的，插入位置在HST基因（At3g11945）的第二个内含子内。将拟南芥HST基因，导入突变体中，能够使突变体性状回复。通过RNAi方法获得HST基因低表达水平的转基因拟南芥植株。
6.通过实时定量PCR和启动子表达分析，得知：该基因在绿色组织中表达量较高，随着叶片的衰老，基因表达量降低。
7.通过ELISA测试，得知：该突变体脱落酸，赤霉素和玉米素水平严重下降，但赤霉素与ABA比值提高；植物生长素（IAA）水平提高。
In this study, we characterized the MsZFN gene from Medicago sativa (alfalfa) and HST gene fromArabidopsis thaliana by gene genetic engineering, transgenic technology and phenotype analysis. Theresults are showed below:
1. A6,829-bp genomic DNA sequence (Access No.: JX131368) was obtained for MsZFN by PCR.Seven exons making up the1,667bp cDNA sequence of MsZFN were interrupted by six introns of820-bp,494-bp,1825-bp,286-bp,641-bp and1093-bp in length when the cDNA and the gDNAsequences were compared.
2. To determine the expression pattern of the MsZFN gene in alfalfa, RT-PCR and qRT-PCR wereperformed. Using RT-PCR, the MsZFN transcript was detected in all alfalfa organs tested, includingroots, stems, leaves, and in florets before and after flower opening. Transcript levels were strongestin leaves compared with stems and roots and was weakest in floral buds and florets. Expressionwas also increased during growth under continuous dark conditions, but not affected by20μMABA,20μM GA3,3%sugar treatments or cold conditions.
3. For transgenic expression experiments, the full-MsZFN coding domain sequence was inserted intothe pCAMBIA1302to generate binary fusion construct35S::MsZFN. To generate transgenicArabidopsis plants expressing MsZFN, Agrobacterium GV3101transformed with plasmid35S::MsZFN was used to infect Arabidopsis and tobacco. The results showed that overexpressionof MsZFN gene delayed flowering time in transgenic plants.
4. We found one Arabidopsis mutant, and the mutant lines are albino and had shorter roots, fewer roothairs, fewer and smaller leaves with shorter petioles, and reduced trichome density. TEM analysisshowed that the mutants had lost the ability to develop into mature chloroplasts. SEM resultsshowed that mutant trichomes were shorter in length and some trichomes had two branches insteadof three. Stomata had bigger openings, suggesting that stomata may close abnormally.
5. The TAIL-PCR and sequencing showed that the mutant phenotypes were caused by a T-DNAinsertion and the insertion was located at the second intron of the Arabidopsis HST gene(At3g11945). When expressed HST gene in mutants, the HST gene could completely rescue themutant phenotype. Transgenic Arabidopsis with low expressed levels of HST gene by RNAitechnology were obtained.
6. To analyze expression pattern of HST gene, qRT-PCR and promoter expression methods wereperformed. The results showed that HST gene was highly expressed in green tissues and the levelsof HST transcript were highest in non-senescent leaves, and the transcript levels graduallydecreased as leaves began to senesce.
7. Enzyme-linked immunosorbent assays (ELISA) were performed to determine concentrations ofdifferent hormones in WT and mutant plants. A substantial decrease in ABA, GA3and ZRconcentrations occurred in mutant plants compared with WT. This disruption of the HST gene also led to a substantial increase (by41.6%) in the IAA content in mutant.
引文
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