大豆脂肪氧化酶基因RNAi表达载体的构建及表达调控的研究
摘要
本项研究应用基因工程手段，克隆大豆脂肪氧化酶基因核心保守序列，构建高效的具有hpRNA和ihpRNA结构的大豆脂肪氧化酶基因RNAi表达载体。通过花粉管通道法将其导入大豆，抑制大豆脂肪氧化酶基因的表达，调控脂肪氧化酶的生物合成过程。以期通过基因工程手段，改变大豆脂肪氧化酶合成途径，降低大豆脂肪氧化酶含量，提高大豆油份含量，培育具有优良品质的大豆品种。取得如下结果：
1．改造植物表达载体pCAMBIA1301，去除其臂内潮酶素基因，并在臂内按逆时针方向添加一个从质粒pBI121上酶切下来的35S启动子。
2．以大豆品种“吉农18号”的基因组DNA为模板，选取大豆三种脂肪氧化酶同功酶基因同源性最高部分，通过PCR扩增得到大豆脂肪氧化酶基因片段，将其克隆到pMD18-Tvector载体上，测序结果表明：克隆片段大小为357bp。对比NCBI基因Bank，与已发表的基因一致。将大豆脂肪氧化酶基因片段按正向+反向的顺序插入到pCAMBIA1301 35S启动子下，构建大豆脂肪氧化酶基因hpRNA干扰表达载体pC1301LoxRi。通过花粉管通道法转化大豆品种“吉农18号”，获得T0代转化籽粒，萌发后以单棵植株叶片的总DNA为模板，以pCAMBIA1301臂内GUS基因设计一对引物进行PCR扩增，结果从6棵植株中扩增得到720bp的特异带，回收该条带连接于pMD18-Tvector载体并测序，结果显示其为要扩增的GUS基因片段。为进一步确定外源基因的整合情况，从PCR阳性植株中随机抽取2株进行Southern blot分析。结果表明，转化株均有杂交带出现，外源基因以单拷贝和双拷贝的形式插入，而未经转化的植株没有杂交信号出现，证明外源基因已整合到大豆基因组中。以转基因大豆籽粒的总RNA反转录成的cDNA为模板做RT-PCR，从电泳条带的亮度上看，转基因植株和非转基因植株的内标18SRNA含量相同，而内源脂肪氧化酶mRNA的含量明显降低。
3．克隆了大豆“吉农18号”自身的一段内含子，片段长度230bp，将其插入到表达载体pC1301LoxRi的正义基因片段和反义基因片段中间，构建大豆脂肪氧化酶基因ihpRNA干扰表达载体pC1301LoxiRi。通过花粉管通道法转化大豆品种“吉农18号”，获得T0代转化籽粒，萌发后以单棵植株叶片的总DNA为模板，以pCAMBIA1301臂内T-DNA区GUS基因设计一对引物进行PCR扩增，结果从18棵植株中扩增得到720bp的特异带，回收该条带连接于pMD18-Tvector载体并测序，结果显示其为要扩增的GUS基因片段。为进一步确定外源基因的整合情况，从PCR阳性植株中随机抽取3株进行Southern blot分析。结果表明，转化株均有杂交带出现，外源基因以单拷贝和双拷贝的形式插入，而未经转化的植株没有杂交信号出现，证明外源基因已整合到大豆基因组中。以转基因大豆籽粒的总RNA反转录成的cDNA为模板做RT-PCR，从电泳条带的亮度上看，转基因植株和非转基因植株的内标18SRNA含量相同，而内源脂肪氧化酶mRNA的含量明显降低。
4．以T1代转化的籽粒为材料，采用紫外分光光度法对脂肪氧化酶活性进行了测定，结果显示，转pC1301LoxRi质粒大豆的脂肪氧化酶活性分别为降低62％，转化pC1301LoxiRi质粒大豆的脂肪氧化酶活性减低74％。说明外源基因的导入有效地抑制了脂肪氧化酶基因的表达。
5．以T1代转化的籽粒为材料，采用SDS-PAGE电泳，结果显示转化pC1301LoxRi质粒大豆的脂肪氧化酶含量比对照平均降低55％，转化pC1301LoxiRi质粒大豆的脂肪氧化酶含量比对照平均降低71％。
6．以T1代转化的籽粒为材料，转基因植株蛋白质的凯氏定氮和索氏提取测定结果显示，转基因植株蛋白质含量均有所下降，脂肪含量均有所上升：其中转化pC1301LoxRi质粒植株蛋白质含量平均为36.06％，比非转基因对照降低0.95个百分点(非转基因蛋白质含量37.01％)，最大降低1.28百分点达到35.73％，脂肪平均含量23.89％，比非转基因对照平均提高了0.74个百分点(非转基因脂肪含量23.15％)，最大提高1.09个百分点，达到24.24％；转pC1301LoxiRi质粒植株蛋白质含量平均为35.63％，比非转基因植株对照降低了1.38个百分点(非转基因蛋白质含量37.01％)，最大降低1.62百分点达到35.39％，脂肪平均含量24.33％，比非转基因对照平均提高了1.18个百分点(非转基因脂肪含量23.15％)，最大提高1.61个百分点，达到24.76％。
7．观察T1代转化植株的主要农艺形状，包括：株高、节数、结荚数、单株粒数、虫食粒、百粒重，发现转基因植株与非转基因对照无明显差别。
8．对转基因植株T2代的PCR分析及PCR-Southern结果证明：外源基因在转基因后代中能遗传。其分离比基本符合孟德尔的遗传规律。
9．探讨了应用重组PCR构建植物基因RNA干扰构件，初步证明其是一种简便、快捷、可行、适用范围广的方法。
10．初步探讨了大豆花粉管通道法的室内转化条件，证明在我国北方地区采用花粉管通道法转化大豆可以在室内进行，每年可以加代一次。
本项研究首次将RNA干扰技术应用于改良大豆脂肪氧化酶含量，取得了良好效果，获得了脂肪氧化酶含量明显降低，脂肪含量明显提高的转基因大豆植株，突破了传统育种方法在改良大豆品质方面易受种质资源限制和育种时间长的缺点，为应用RNA干扰技术改良大豆品质提高大豆油份含量探索了新的途径，实现了大豆品质改良育种和高油育种的方法创新，也为以后通过RNA干扰技术改良大豆其它营养抑制因子，提高大豆品质奠定了基础，具有重要的理论和实践意义。
The purpose of this research is to decrease the content of lipoxidase in soybean and increasethe content of soybean oil, hope to found some new species that have excellence grace. Withgenetic engineering method, the lipoxidase gene of core consensus sequence were cloned, ahighly efficient with hpRNA and ihpRNA of RNAi expression system were constructed. Thenthe gene constructs were introduced into soybean mediated by pollen tube passage method inorder to restrain the gene expression of lipoxidase and regulate the biological composing processof lipoxidase. The main results are as fellow:
1. Transform the plant expression vector pCAMBIA1301, removal the hygromycin, and add35s promoter according to anti-clocwise from plasmid pBI121.
2. Using the genomic DNA of soybean variety "JiNong 18" as template, to select the highesthomology from the three lipoxidase of isozyme. The lipoxidase was isolated by PCR and wascloned into pMD18-T Vector. The size of this promoter is 357bp. Sequencing analysis of thislipoxidase gene through NCBI GENE BANK showed that was the same to the enunciatedbefore. Using the fragment of soybean lipoxidase gene into pCAMBIA1301 35S promoter, thefragment is norientation and inversus, then, pC1301LoxRi, the hpRNAi expression vector ofsoybean lipoxidase gene was constructed. The To seeds of soybean variety "JiNong18" wasreapped by pollen tube passage method. After the seeds were germinated, using geromic DNA ofthe regenerated plants' leaves as template, PCR amplification was performed withpCAMBIA1301 gus gene primers. The 721bp PCR product was obtained respectively from the12 transgenic plants. Link the extraction of the product to pMD18-Tvector, sequencing analysisshowed the fragment amplified was the gus gene we need. Then, Southern blot analysis wasperformed to further identify the number of copies that of gene integrated into the geromic DNAof the transgenic plant, And the result indicates that hybrid signal occurred in both the transgenicplant lines with one or two copies, respectively. But there was no hybrid signals shown in plantsthat hadn't been transplanted. Using the cDNA from the transgene of soybean as template forRT-PCR, the content of the lipoxidase mRNA in transgene plants decreased obviously.
3. Clon a piece of intervening sequence from soybean variety "JiNongl8", the size of thispromoter is 230 bp. The fragment of soybean lipoxidase gene into pC1301LoxRi expressionvector is between the the norientation and inversus. Then the hpRNAi expression vector ofsoybean lipoxidase gene pC1301LoxiRi was constructed. The T_0 seeds of soybean variety "JiNong18" was reapped by pollen tube passage method. After the seeds were germinated, usinggeromic DNA of the regenerated plants' leaves as template, PCR amplification was performedwith pCAMBIA1301 gus gene primers. The 721bp PCR product was obtained respectively fromthe 14 transgenic plants. Link the extraction of the product to pMD18-Tvector, sequencinganalysis showed the fragment amplified was the gus gene we need. Then, Southern blot analysiswas performed to further identify the number of copies that of gene integrated into the geromicDNA of the transgenic plant, And the result indicates that hybrid signal occurred in both thetransgenic plant lines with one or two copies, respectively. But there was no hybrid signalsshown in plants that hadn't been transplanted. Using the cDNA from the transgene of soybean astemplate for fRT-PCR, the content of the lipoxidase mRNA in transgene plants decreasedobviously.
4. From the generation transgenic T_1 seeds, the activity of lipoxidase was assayed using theuntraviolet spectrophotometry method. The result indicates that the activity of soybeanlipoxidase which transformed by pC1301LoxRi plasmid decreased by 62%, and the activity ofsoybean lipoxidase which transformed by pC1301LoxiRi plasmid decreased 76%. And theactivity indicates that the insert of introduced has efficiently restrained the expression oflipoxidase.
5.The content of lipoxidase also assayed from T_1 generation transgenic soybean usingSDS-PAGE method to test. The result indicate that compare to control, the content of lipoxidaseof transgenic soybean were decreased, among the total which transformed by pC1301LoxRiplasmid average decreased by 55%, and the activity of soybean lipoxidase by pC1301LoxiRiplasmid average decreased 71%.
6. Use the transgenic T1 seeds, The protein and oil contents in the seeds of the transgenic soybeans weredetermined by the Kjeldahl method for protein content and the SoxMet method for oil content, respectively.The results showed that the content of protein of transgenic soybean were decreased, among the total,average content of protein which transformed by pC1301LoxRi plasmid were 36.06%,and thenon-transgenic decrease 0.95 persent, the maximum decrease 1.28 persent which to achieve35.73%; the average content of oil which transformed by pC1301LoxiRi plasmid were 23.89%,and the non-transgenic increase 0.74 percent, the maximum increase 1.09 percent which toachieve 24.24%; the average content of protein which transformed by pC1301LoxiRi plasmidwere 35.63%, and the non-transgenic decrease 1.38 percent, the maximum decrease 1.62 percentwhich to achieve 35.39%; the average content of oil which transformed by pC1301LoxiRiplasmid were 24.33%, and the non-transgenic increase 1.18 percent, the maximum increase1.61 percent which to achieve 24.76%.
7. The result shows that the agronomy character of the transgenic T1 soybean has no obviousdifference with the control through plant laboratory observing which include plant height, thenode number, pod numbers, grain number of single plant, the seeds eaten, 100-grain weight.
8. The transgenic T_2 seeds were determined by PCR and PCR-Southern blot, the result showsthat the foreign gene may be heridable over successive generations, the segregation ratio wasbasically corresponds with the Mendelian genetic law.
9. Construct the RNA components of plant gene applied recombinant PCR was discussed, itpresents preliminary proof that it is a simple, rapid, feasible and wide application range method.
10. Indoor conversion condition of pollen tube passage was discussed, it presents that it cancarry out convert soybean indoor by pollen tube passage in the northern part of the country,adding generation one time every year.
The study has obtained the good effect and obtained the transgenic soybean whichlipoxidase content obviously decrease and the fat content obviously increase through applyingfor the RNA interference technology to the improvement soybean lipoxidase content at the firsttime, it broked through the shortcoming of the traditional breeding method in the improvementsoybean quality aspect easily the idioplasm resourced limit and the breeding time long, itexplored the new way for improvement soybean quality and enhanced the soybean oil contentusing the RNA inference technology, it has realized the innovation of the soybean qualityimprovement breeding and the high oil breeding method, also has the important theory and thepractice significance. For later through the RNA interference technology improvement soybeanother nutrition inhibiting factors, and lay the foundation for enhance the soybean quality.
1．改造植物表达载体pCAMBIA1301，去除其臂内潮酶素基因，并在臂内按逆时针方向添加一个从质粒pBI121上酶切下来的35S启动子。
2．以大豆品种“吉农18号”的基因组DNA为模板，选取大豆三种脂肪氧化酶同功酶基因同源性最高部分，通过PCR扩增得到大豆脂肪氧化酶基因片段，将其克隆到pMD18-Tvector载体上，测序结果表明：克隆片段大小为357bp。对比NCBI基因Bank，与已发表的基因一致。将大豆脂肪氧化酶基因片段按正向+反向的顺序插入到pCAMBIA1301 35S启动子下，构建大豆脂肪氧化酶基因hpRNA干扰表达载体pC1301LoxRi。通过花粉管通道法转化大豆品种“吉农18号”，获得T0代转化籽粒，萌发后以单棵植株叶片的总DNA为模板，以pCAMBIA1301臂内GUS基因设计一对引物进行PCR扩增，结果从6棵植株中扩增得到720bp的特异带，回收该条带连接于pMD18-Tvector载体并测序，结果显示其为要扩增的GUS基因片段。为进一步确定外源基因的整合情况，从PCR阳性植株中随机抽取2株进行Southern blot分析。结果表明，转化株均有杂交带出现，外源基因以单拷贝和双拷贝的形式插入，而未经转化的植株没有杂交信号出现，证明外源基因已整合到大豆基因组中。以转基因大豆籽粒的总RNA反转录成的cDNA为模板做RT-PCR，从电泳条带的亮度上看，转基因植株和非转基因植株的内标18SRNA含量相同，而内源脂肪氧化酶mRNA的含量明显降低。
3．克隆了大豆“吉农18号”自身的一段内含子，片段长度230bp，将其插入到表达载体pC1301LoxRi的正义基因片段和反义基因片段中间，构建大豆脂肪氧化酶基因ihpRNA干扰表达载体pC1301LoxiRi。通过花粉管通道法转化大豆品种“吉农18号”，获得T0代转化籽粒，萌发后以单棵植株叶片的总DNA为模板，以pCAMBIA1301臂内T-DNA区GUS基因设计一对引物进行PCR扩增，结果从18棵植株中扩增得到720bp的特异带，回收该条带连接于pMD18-Tvector载体并测序，结果显示其为要扩增的GUS基因片段。为进一步确定外源基因的整合情况，从PCR阳性植株中随机抽取3株进行Southern blot分析。结果表明，转化株均有杂交带出现，外源基因以单拷贝和双拷贝的形式插入，而未经转化的植株没有杂交信号出现，证明外源基因已整合到大豆基因组中。以转基因大豆籽粒的总RNA反转录成的cDNA为模板做RT-PCR，从电泳条带的亮度上看，转基因植株和非转基因植株的内标18SRNA含量相同，而内源脂肪氧化酶mRNA的含量明显降低。
4．以T1代转化的籽粒为材料，采用紫外分光光度法对脂肪氧化酶活性进行了测定，结果显示，转pC1301LoxRi质粒大豆的脂肪氧化酶活性分别为降低62％，转化pC1301LoxiRi质粒大豆的脂肪氧化酶活性减低74％。说明外源基因的导入有效地抑制了脂肪氧化酶基因的表达。
5．以T1代转化的籽粒为材料，采用SDS-PAGE电泳，结果显示转化pC1301LoxRi质粒大豆的脂肪氧化酶含量比对照平均降低55％，转化pC1301LoxiRi质粒大豆的脂肪氧化酶含量比对照平均降低71％。
6．以T1代转化的籽粒为材料，转基因植株蛋白质的凯氏定氮和索氏提取测定结果显示，转基因植株蛋白质含量均有所下降，脂肪含量均有所上升：其中转化pC1301LoxRi质粒植株蛋白质含量平均为36.06％，比非转基因对照降低0.95个百分点(非转基因蛋白质含量37.01％)，最大降低1.28百分点达到35.73％，脂肪平均含量23.89％，比非转基因对照平均提高了0.74个百分点(非转基因脂肪含量23.15％)，最大提高1.09个百分点，达到24.24％；转pC1301LoxiRi质粒植株蛋白质含量平均为35.63％，比非转基因植株对照降低了1.38个百分点(非转基因蛋白质含量37.01％)，最大降低1.62百分点达到35.39％，脂肪平均含量24.33％，比非转基因对照平均提高了1.18个百分点(非转基因脂肪含量23.15％)，最大提高1.61个百分点，达到24.76％。
7．观察T1代转化植株的主要农艺形状，包括：株高、节数、结荚数、单株粒数、虫食粒、百粒重，发现转基因植株与非转基因对照无明显差别。
8．对转基因植株T2代的PCR分析及PCR-Southern结果证明：外源基因在转基因后代中能遗传。其分离比基本符合孟德尔的遗传规律。
9．探讨了应用重组PCR构建植物基因RNA干扰构件，初步证明其是一种简便、快捷、可行、适用范围广的方法。
10．初步探讨了大豆花粉管通道法的室内转化条件，证明在我国北方地区采用花粉管通道法转化大豆可以在室内进行，每年可以加代一次。
本项研究首次将RNA干扰技术应用于改良大豆脂肪氧化酶含量，取得了良好效果，获得了脂肪氧化酶含量明显降低，脂肪含量明显提高的转基因大豆植株，突破了传统育种方法在改良大豆品质方面易受种质资源限制和育种时间长的缺点，为应用RNA干扰技术改良大豆品质提高大豆油份含量探索了新的途径，实现了大豆品质改良育种和高油育种的方法创新，也为以后通过RNA干扰技术改良大豆其它营养抑制因子，提高大豆品质奠定了基础，具有重要的理论和实践意义。
The purpose of this research is to decrease the content of lipoxidase in soybean and increasethe content of soybean oil, hope to found some new species that have excellence grace. Withgenetic engineering method, the lipoxidase gene of core consensus sequence were cloned, ahighly efficient with hpRNA and ihpRNA of RNAi expression system were constructed. Thenthe gene constructs were introduced into soybean mediated by pollen tube passage method inorder to restrain the gene expression of lipoxidase and regulate the biological composing processof lipoxidase. The main results are as fellow:
1. Transform the plant expression vector pCAMBIA1301, removal the hygromycin, and add35s promoter according to anti-clocwise from plasmid pBI121.
2. Using the genomic DNA of soybean variety "JiNong 18" as template, to select the highesthomology from the three lipoxidase of isozyme. The lipoxidase was isolated by PCR and wascloned into pMD18-T Vector. The size of this promoter is 357bp. Sequencing analysis of thislipoxidase gene through NCBI GENE BANK showed that was the same to the enunciatedbefore. Using the fragment of soybean lipoxidase gene into pCAMBIA1301 35S promoter, thefragment is norientation and inversus, then, pC1301LoxRi, the hpRNAi expression vector ofsoybean lipoxidase gene was constructed. The To seeds of soybean variety "JiNong18" wasreapped by pollen tube passage method. After the seeds were germinated, using geromic DNA ofthe regenerated plants' leaves as template, PCR amplification was performed withpCAMBIA1301 gus gene primers. The 721bp PCR product was obtained respectively from the12 transgenic plants. Link the extraction of the product to pMD18-Tvector, sequencing analysisshowed the fragment amplified was the gus gene we need. Then, Southern blot analysis wasperformed to further identify the number of copies that of gene integrated into the geromic DNAof the transgenic plant, And the result indicates that hybrid signal occurred in both the transgenicplant lines with one or two copies, respectively. But there was no hybrid signals shown in plantsthat hadn't been transplanted. Using the cDNA from the transgene of soybean as template forRT-PCR, the content of the lipoxidase mRNA in transgene plants decreased obviously.
3. Clon a piece of intervening sequence from soybean variety "JiNongl8", the size of thispromoter is 230 bp. The fragment of soybean lipoxidase gene into pC1301LoxRi expressionvector is between the the norientation and inversus. Then the hpRNAi expression vector ofsoybean lipoxidase gene pC1301LoxiRi was constructed. The T_0 seeds of soybean variety "JiNong18" was reapped by pollen tube passage method. After the seeds were germinated, usinggeromic DNA of the regenerated plants' leaves as template, PCR amplification was performedwith pCAMBIA1301 gus gene primers. The 721bp PCR product was obtained respectively fromthe 14 transgenic plants. Link the extraction of the product to pMD18-Tvector, sequencinganalysis showed the fragment amplified was the gus gene we need. Then, Southern blot analysiswas performed to further identify the number of copies that of gene integrated into the geromicDNA of the transgenic plant, And the result indicates that hybrid signal occurred in both thetransgenic plant lines with one or two copies, respectively. But there was no hybrid signalsshown in plants that hadn't been transplanted. Using the cDNA from the transgene of soybean astemplate for fRT-PCR, the content of the lipoxidase mRNA in transgene plants decreasedobviously.
4. From the generation transgenic T_1 seeds, the activity of lipoxidase was assayed using theuntraviolet spectrophotometry method. The result indicates that the activity of soybeanlipoxidase which transformed by pC1301LoxRi plasmid decreased by 62%, and the activity ofsoybean lipoxidase which transformed by pC1301LoxiRi plasmid decreased 76%. And theactivity indicates that the insert of introduced has efficiently restrained the expression oflipoxidase.
5.The content of lipoxidase also assayed from T_1 generation transgenic soybean usingSDS-PAGE method to test. The result indicate that compare to control, the content of lipoxidaseof transgenic soybean were decreased, among the total which transformed by pC1301LoxRiplasmid average decreased by 55%, and the activity of soybean lipoxidase by pC1301LoxiRiplasmid average decreased 71%.
6. Use the transgenic T1 seeds, The protein and oil contents in the seeds of the transgenic soybeans weredetermined by the Kjeldahl method for protein content and the SoxMet method for oil content, respectively.The results showed that the content of protein of transgenic soybean were decreased, among the total,average content of protein which transformed by pC1301LoxRi plasmid were 36.06%,and thenon-transgenic decrease 0.95 persent, the maximum decrease 1.28 persent which to achieve35.73%; the average content of oil which transformed by pC1301LoxiRi plasmid were 23.89%,and the non-transgenic increase 0.74 percent, the maximum increase 1.09 percent which toachieve 24.24%; the average content of protein which transformed by pC1301LoxiRi plasmidwere 35.63%, and the non-transgenic decrease 1.38 percent, the maximum decrease 1.62 percentwhich to achieve 35.39%; the average content of oil which transformed by pC1301LoxiRiplasmid were 24.33%, and the non-transgenic increase 1.18 percent, the maximum increase1.61 percent which to achieve 24.76%.
7. The result shows that the agronomy character of the transgenic T1 soybean has no obviousdifference with the control through plant laboratory observing which include plant height, thenode number, pod numbers, grain number of single plant, the seeds eaten, 100-grain weight.
8. The transgenic T_2 seeds were determined by PCR and PCR-Southern blot, the result showsthat the foreign gene may be heridable over successive generations, the segregation ratio wasbasically corresponds with the Mendelian genetic law.
9. Construct the RNA components of plant gene applied recombinant PCR was discussed, itpresents preliminary proof that it is a simple, rapid, feasible and wide application range method.
10. Indoor conversion condition of pollen tube passage was discussed, it presents that it cancarry out convert soybean indoor by pollen tube passage in the northern part of the country,adding generation one time every year.
The study has obtained the good effect and obtained the transgenic soybean whichlipoxidase content obviously decrease and the fat content obviously increase through applyingfor the RNA interference technology to the improvement soybean lipoxidase content at the firsttime, it broked through the shortcoming of the traditional breeding method in the improvementsoybean quality aspect easily the idioplasm resourced limit and the breeding time long, itexplored the new way for improvement soybean quality and enhanced the soybean oil contentusing the RNA inference technology, it has realized the innovation of the soybean qualityimprovement breeding and the high oil breeding method, also has the important theory and thepractice significance. For later through the RNA interference technology improvement soybeanother nutrition inhibiting factors, and lay the foundation for enhance the soybean quality.
引文
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