柑橘转GFP、AP1和LFY基因植株群体的分子鉴定与基因表达研究
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摘要
转基因技术应用于改良农作物的特定性状,如耐除草剂、抗病性、抗虫性、抗环境胁迫、提高产量、改善品质等。还可以通过转基因植株超量或抑制特定基因的表达进行目的基因的功能验证。转基因植株能否广泛应用,其关键在于所转基因能否在遗传转化体内稳定整合和表达。本研究以转GFP、AP1、LFY柑橘为材料,分析外源基因的拷贝数、T-DNA整合、甲基化和外源基因表达之间的相关性；采用Real-time PCR在转基因桠柑中筛选单拷贝的转基因植株,以验证Real-time PCR在拷贝数分析上的可行性；分析转AP1和LFY柑橘的外源基因拷贝数和表达,研究早花转基因植株外源AP1或LFY及内源开花基因的表达。主要结果如下：
1. Southern杂交分析118棵转基因植株外源基因的拷贝数。结果表明大部分转基因植株拷贝数为1-2个。转GFP基因8株冰糖橙中5株为多拷贝(拷贝数大于3),转AP1桠柑(43株)有超过一半植株为多拷贝；转GFP佛罗斯特脐橙、转AP1茶枝柑、转LFY改良橙与尾张有性杂种、转LFY冰糖橙的拷贝数均为1-2个；转GFP改良橙与尾张有性杂种和转AP1金柑各有一个株系为多拷贝,其它为1-2个拷贝。不同转基因柑橘株系中都有T-DNA整合缺失的出现,转AP1茶枝柑、转LFY冰糖橙各有一个株系的GUS基因缺失。
2.转GFP柑橘荧光检测。转GFP基因佛罗斯特脐橙愈伤荧光稳定表达,在愈伤分化旺盛时绿色荧光表达较强。转GFP植株叶片荧光检测发现大部分转基因植株荧光正常表达,嫩叶荧光比老叶强；改良橙与尾张有性杂种有一个转基因株系荧光未检测到,即发生转基因沉默；一个株系的转基因冰糖橙荧光呈现斑驳化。
3. Southern杂交和反向PCR分析转GFP柑橘的T-DNA整合情况。详细分析3个转GFP改良橙与尾张有性杂种株系,6个转GFP冰糖橙的T-DNA整合：在6棵多拷贝的转GFP植株中,4棵植株中有T-DNA正向重复的排列；3株转基因冰糖橙都有一个T-DNA正向重复,1株转基因沉默的改良橙与尾张有性杂种中发现至少两个T-DNA重复；进一步测序发现T-DNA重复的左右边界序列都有不同程度的缺失,而转基因的改良橙与尾张有性杂种的两个T-DNA重复中存在一个500 bp片段的缺失,缺失部分为Mos-pro和部分nptII序列。
4.亚硫酸氢盐测序法分析转GFP柑橘的nptⅡ、GFP和CaMV35S启动子的甲基化程度。结果显示1-2个拷贝的转GFP柑橘外源基因和35S甲基化和质粒DNA甲基化程度类似；多拷贝转基因改良橙与尾张有性杂种其nptII序列有10%-20%的胞嘧啶甲基化,而该转基因植株的GFP和35S中胞嘧啶甲基化程度高达70%-90%,特别是CG和CNG类型的对称式的甲基化程度为90%。多拷贝转基因冰糖橙nptlI,GFP和CaMV 35S启动子序列的甲基化分别为10%-40%,20%-60%和60%-80%。
5.转基因柑橘外源基因表达。Northern杂交和Real-time RT-PCR分析nptII和GFP基因的表达,不同拷贝之间nptII表达没有差异,GFP在大多数多拷贝植株表达较低,转基因沉默植株GFP表达非常低；转LFY的改良橙与尾张有性杂种和冰糖橙的外源基因表达与拷贝数也没有相关性。
6.Real-time PCR在转AP1桠柑中筛选单拷贝植株。Real-time PCR分析结果表明43个转基因桠柑中有11株单拷贝,与Southern结果一致。Real-time PCR法检测的相对拷贝数与Southern杂交结果的相关系数分别为：AP1,0.7521；GUS,0.5856;nptlI,0.8222。
7.转AP1或LFY柑橘开花情况。5株转AP1金柑植株中1株移栽温室11个月开花(段艳欣,2006),在随后两年均正常开花；另有2株第二年开花,随后一年也正常开花；第三年,一个转基因株系表现为比对照植株晚花,其它转化或未转化植株均开花。3个转LFY的改良橙与尾张有性杂种2个株系早花移栽温室一年后开花;8个转LFY冰糖橙1株早花移栽温室两年后开花；其它转化和未转化植株都没开花。
8.外源开花基因和内源开花基因的表达。转AP1金柑的不同开花植株中外源AP1表达各有差异,部分开花植株的内源CiLFY上调表达,而内源CiAP1在开花植株中下调表达。在转LFY的冰糖橙和改良橙与尾张有性杂种中,早花植株的外源LFY表达相对较高,大部分转基因植株表现为内源CiAP1上调表达和CiLFY下调表达。早花转基因金柑可能与CiFT、CiLFY的上调表达和CiTFL1的下调表达有关,晚花与CiAP1和CiTFL1上调表达和CiFT的下调表达相关。转LFY的冰糖橙和改良橙与尾张有性杂种的早花转基因株系都表现为CiFT、CiWUS和CiTFL1下调表达。其它内源开花基因在转基因植株间没有规律性的变化。以上结果表明CiTFL1下调表达在调控转AP1或LFY柑橘开花时间上起着决定性的作用。
本文还对外源基因拷贝数、T-DNA重复、启动子与外源基因甲基化对外源基因表达的影响,Real-time PCR分析拷贝数的可行性以及外源AP1和LFY与内源开花基因在调控花期中的相互作用进行了讨论。
Transgene technology could be applied to improve certain traits of crops such as herbicide, disease, insect or environmental stress-resistance, product yeild and quality improvement. Extensive application of transgenic plants depends on the integration of the exogenous gene into plant genome and its expression stability. In this study, GFP transgenic citrus plants were analyzed to characterize the correlation of exogenous gene expression with its copy number, the integration of T-DNA and methylation of transgene as well as its promoter. Real-time PCR was validated to be feasible on gene copy number analysis and applied to screen transgenic Ponkan(Citrus reticulata cv. Ponkan) plants of single copy. The transgene copy number and expression were analyzed in API or LFY transgenic citrus plants. Expression of exogenous API, LFY and endogenous flowering related genes were studied in early-flowing transgenic plants. The main results were listed as follows:
1. Southern blot of 118 transgenic citrus to determine transgene copy number. Most transgenic plants got 1-2 copies of tansgene. Five of the eight GFP transgenic Bingtangcheng (C. sinensis cv. Bingtangcheng) had multiple copies (more than three) of exogenous gene; More than half of the API transgenic Ponkan (43 plants) had multiple copies of tansgene. The GFP transgenic Florest navel orange (C. sinensis cv. Florest), API transgenic Chazhigan tangerine, LFY transgenic GWZ (C. sinensis cv. GailiangchengxC. unshui), LFY transgenic Bingtangcheng all had copy number of 1-2. GFP transgenic GWZ and API transgenic 'Meiwa' kumquat(Fortunella crassifolia) each had one line with multiple copies of exogenous gene, while others had 1-2 copies. Truncated T-DNA inserts was observed in different transgenic citrus, absence of GUS was found in one line of API transgenic Chazhigan tangerine or LFY transgenic Bingtangcheng.
2. GFP fluorescence detection of transgenic plants under stereomicroscope. The GFP expression was stable and bright in transgenic Florest navel orange calluses, brighter at activated differentiation state. GFP fluorescence was normally expressed in leaves of most transgenic plants; young leaves got stronger fluorescence than old ones. In one line of GFP transgenic GWZ, GFP fluorescence was not observed, indicating transgene silencing occured. One line of GFP transgenic Bingtangcheng showed mottled fluorescence.
3 T-DNA integration analysis of GFP transgenic citrus was analyzed by Southern blot and inverse PCR. Detailed analysis of 3 GFP transgenic GWZ and 6 GFP transgenic Bingtangcheng revealed that four of the six GFP transgenic plants with multiple copies exhibited T-DNA direct repeats; each of three GFP transgenic Bingtangcheng had one T-DNA direct repeat, at least two T-DNA direct repeats were found in the GFP transgenic GWZ lines with multiple copies and transgene silencing. Truncated left border (LB) and right border (RB) was observed in T-DNA repeats by further sequencing. One of the two T-DNA repeats in GFP transgenic silencing GWZ was truncated 500 bp sequence, containing Nos-pro and part nptⅡ.
4 Methylation of nptⅡ, GFP and 35S promoter in GFP transgenic citrus was analyzed by bisulfite sequencing. The methylation with GFP, nptⅡand 35S of 1-2 copies transgenic plants was similar with plasmid.10%-20% cytosines methylation of nptⅡwas found in the ransgenic GWZ line with multiple copies, while for GFP and 35S promoter were 70%-90%, in which symmetrical methylation of CG and CNG reached 90%. Cytosines methylation degree of nptⅡin multiple copies transgenic Bingtangcheng line was 10%-40%, GFP and 35S promoter in these lines were 20%-60% and 60%-80%, respectively.
5. Expression of exogenous genes in transgenic citrus. Expression of GFP and nptⅡanalysis by Northern blot and Real-time RT-PCR showed no difference on nptⅡexpression among transgenic plants with different copy transgene numbers. GFP was low expressed in most of the multiple copies transgenic plants. Especially, GFP showed extremely low transcriptional level in transgene silenced plants. There was no correlation between the expression of transgene and copy number in LFY transgenic GWZ or Bingtangcheng.
6 Real-time PCR was applied to screen transgenic Ponkan with single copy. The result indicated that 11 of 43 AP1 transgenic Ponkan had single copy, consistent with the result of Southern blot. The correlation coefficient between results of Real-time PCR and Southern blot of API, GUS and nptⅡwas 0.7521,0.5856 and 0.8222, respectively.
7 Flowering of AP1 or LFY transgenic citrusplants also were investigated. One of the five AP1 transgenic 'Meiwa' kumquat started flowering 11 months after transfer to greenhouse (Duan,2006) and continued to flower in the next two years, other two transgenic plants and three of seven non-transformed plants flowered after 23 months and continued to flower in the next year, In the third year, one transgenic plants delayed flowering when other transgenic or no transgenic plants all flowered. Two of the three LFY transgenic GWZ started flowering one year after transfer to greenhouse. One of the eight LFY transgenic Bingtangcheng was early flowering after transfer to greenhouse two years. Other transgenic or non-transgenic plants did not flower.
8 Expression of exogenous AP1, LFY and endogenous flowering related genes. The expression of exogenous AP1 was different among AP1 transgenic 'Meiwa' kumquat which flowered. CiLFY was up-regulated in some of the flowering transgenic plants, while CiAP1 was down-regulated. In LFY transgenic GWZ and Bingtangcheng, the expression of exogenous LFY was higher in early flowering transgenic plants. Endogenous CiAPl up-regulation and CiLFY down-regulation were found in most LFY transgenic plants. Endogenous CiLFY and CiFT up-regulation, CiTFL1 down-regulation may be related to early flowering of AP1 transgenic 'Meiwa' kumquat. CiAP1 and CiTFL1 up-regulation, CiFT down-regulation may be assosiated with delay flowering of AP1 transgenic 'Meiwa' kumquat. CiFT, CiWUS and CiTFL1 were down-regulated in all early flowering LFY transgenic GWZ and Bingtangcheng. The results indicated that down-regulated of CiTFL1 play a decisive role in flowering time regulation of transgenic AP1 or LFY citrus.
In this study, impact on transgene expression by exogenous gene copy number, T-DNA repeat, methylation of tansgene and its promoter was explored. Additionaly, feasibility of Real-time PCR on gene copy number analysis, the interaction of AP1 or LFY and endogenous flowering genes in controlling juvenile phase were discussed.
1. Southern杂交分析118棵转基因植株外源基因的拷贝数。结果表明大部分转基因植株拷贝数为1-2个。转GFP基因8株冰糖橙中5株为多拷贝(拷贝数大于3),转AP1桠柑(43株)有超过一半植株为多拷贝；转GFP佛罗斯特脐橙、转AP1茶枝柑、转LFY改良橙与尾张有性杂种、转LFY冰糖橙的拷贝数均为1-2个；转GFP改良橙与尾张有性杂种和转AP1金柑各有一个株系为多拷贝,其它为1-2个拷贝。不同转基因柑橘株系中都有T-DNA整合缺失的出现,转AP1茶枝柑、转LFY冰糖橙各有一个株系的GUS基因缺失。
2.转GFP柑橘荧光检测。转GFP基因佛罗斯特脐橙愈伤荧光稳定表达,在愈伤分化旺盛时绿色荧光表达较强。转GFP植株叶片荧光检测发现大部分转基因植株荧光正常表达,嫩叶荧光比老叶强；改良橙与尾张有性杂种有一个转基因株系荧光未检测到,即发生转基因沉默；一个株系的转基因冰糖橙荧光呈现斑驳化。
3. Southern杂交和反向PCR分析转GFP柑橘的T-DNA整合情况。详细分析3个转GFP改良橙与尾张有性杂种株系,6个转GFP冰糖橙的T-DNA整合：在6棵多拷贝的转GFP植株中,4棵植株中有T-DNA正向重复的排列；3株转基因冰糖橙都有一个T-DNA正向重复,1株转基因沉默的改良橙与尾张有性杂种中发现至少两个T-DNA重复；进一步测序发现T-DNA重复的左右边界序列都有不同程度的缺失,而转基因的改良橙与尾张有性杂种的两个T-DNA重复中存在一个500 bp片段的缺失,缺失部分为Mos-pro和部分nptII序列。
4.亚硫酸氢盐测序法分析转GFP柑橘的nptⅡ、GFP和CaMV35S启动子的甲基化程度。结果显示1-2个拷贝的转GFP柑橘外源基因和35S甲基化和质粒DNA甲基化程度类似；多拷贝转基因改良橙与尾张有性杂种其nptII序列有10%-20%的胞嘧啶甲基化,而该转基因植株的GFP和35S中胞嘧啶甲基化程度高达70%-90%,特别是CG和CNG类型的对称式的甲基化程度为90%。多拷贝转基因冰糖橙nptlI,GFP和CaMV 35S启动子序列的甲基化分别为10%-40%,20%-60%和60%-80%。
5.转基因柑橘外源基因表达。Northern杂交和Real-time RT-PCR分析nptII和GFP基因的表达,不同拷贝之间nptII表达没有差异,GFP在大多数多拷贝植株表达较低,转基因沉默植株GFP表达非常低；转LFY的改良橙与尾张有性杂种和冰糖橙的外源基因表达与拷贝数也没有相关性。
6.Real-time PCR在转AP1桠柑中筛选单拷贝植株。Real-time PCR分析结果表明43个转基因桠柑中有11株单拷贝,与Southern结果一致。Real-time PCR法检测的相对拷贝数与Southern杂交结果的相关系数分别为：AP1,0.7521；GUS,0.5856;nptlI,0.8222。
7.转AP1或LFY柑橘开花情况。5株转AP1金柑植株中1株移栽温室11个月开花(段艳欣,2006),在随后两年均正常开花；另有2株第二年开花,随后一年也正常开花；第三年,一个转基因株系表现为比对照植株晚花,其它转化或未转化植株均开花。3个转LFY的改良橙与尾张有性杂种2个株系早花移栽温室一年后开花;8个转LFY冰糖橙1株早花移栽温室两年后开花；其它转化和未转化植株都没开花。
8.外源开花基因和内源开花基因的表达。转AP1金柑的不同开花植株中外源AP1表达各有差异,部分开花植株的内源CiLFY上调表达,而内源CiAP1在开花植株中下调表达。在转LFY的冰糖橙和改良橙与尾张有性杂种中,早花植株的外源LFY表达相对较高,大部分转基因植株表现为内源CiAP1上调表达和CiLFY下调表达。早花转基因金柑可能与CiFT、CiLFY的上调表达和CiTFL1的下调表达有关,晚花与CiAP1和CiTFL1上调表达和CiFT的下调表达相关。转LFY的冰糖橙和改良橙与尾张有性杂种的早花转基因株系都表现为CiFT、CiWUS和CiTFL1下调表达。其它内源开花基因在转基因植株间没有规律性的变化。以上结果表明CiTFL1下调表达在调控转AP1或LFY柑橘开花时间上起着决定性的作用。
本文还对外源基因拷贝数、T-DNA重复、启动子与外源基因甲基化对外源基因表达的影响,Real-time PCR分析拷贝数的可行性以及外源AP1和LFY与内源开花基因在调控花期中的相互作用进行了讨论。
Transgene technology could be applied to improve certain traits of crops such as herbicide, disease, insect or environmental stress-resistance, product yeild and quality improvement. Extensive application of transgenic plants depends on the integration of the exogenous gene into plant genome and its expression stability. In this study, GFP transgenic citrus plants were analyzed to characterize the correlation of exogenous gene expression with its copy number, the integration of T-DNA and methylation of transgene as well as its promoter. Real-time PCR was validated to be feasible on gene copy number analysis and applied to screen transgenic Ponkan(Citrus reticulata cv. Ponkan) plants of single copy. The transgene copy number and expression were analyzed in API or LFY transgenic citrus plants. Expression of exogenous API, LFY and endogenous flowering related genes were studied in early-flowing transgenic plants. The main results were listed as follows:
1. Southern blot of 118 transgenic citrus to determine transgene copy number. Most transgenic plants got 1-2 copies of tansgene. Five of the eight GFP transgenic Bingtangcheng (C. sinensis cv. Bingtangcheng) had multiple copies (more than three) of exogenous gene; More than half of the API transgenic Ponkan (43 plants) had multiple copies of tansgene. The GFP transgenic Florest navel orange (C. sinensis cv. Florest), API transgenic Chazhigan tangerine, LFY transgenic GWZ (C. sinensis cv. GailiangchengxC. unshui), LFY transgenic Bingtangcheng all had copy number of 1-2. GFP transgenic GWZ and API transgenic 'Meiwa' kumquat(Fortunella crassifolia) each had one line with multiple copies of exogenous gene, while others had 1-2 copies. Truncated T-DNA inserts was observed in different transgenic citrus, absence of GUS was found in one line of API transgenic Chazhigan tangerine or LFY transgenic Bingtangcheng.
2. GFP fluorescence detection of transgenic plants under stereomicroscope. The GFP expression was stable and bright in transgenic Florest navel orange calluses, brighter at activated differentiation state. GFP fluorescence was normally expressed in leaves of most transgenic plants; young leaves got stronger fluorescence than old ones. In one line of GFP transgenic GWZ, GFP fluorescence was not observed, indicating transgene silencing occured. One line of GFP transgenic Bingtangcheng showed mottled fluorescence.
3 T-DNA integration analysis of GFP transgenic citrus was analyzed by Southern blot and inverse PCR. Detailed analysis of 3 GFP transgenic GWZ and 6 GFP transgenic Bingtangcheng revealed that four of the six GFP transgenic plants with multiple copies exhibited T-DNA direct repeats; each of three GFP transgenic Bingtangcheng had one T-DNA direct repeat, at least two T-DNA direct repeats were found in the GFP transgenic GWZ lines with multiple copies and transgene silencing. Truncated left border (LB) and right border (RB) was observed in T-DNA repeats by further sequencing. One of the two T-DNA repeats in GFP transgenic silencing GWZ was truncated 500 bp sequence, containing Nos-pro and part nptⅡ.
4 Methylation of nptⅡ, GFP and 35S promoter in GFP transgenic citrus was analyzed by bisulfite sequencing. The methylation with GFP, nptⅡand 35S of 1-2 copies transgenic plants was similar with plasmid.10%-20% cytosines methylation of nptⅡwas found in the ransgenic GWZ line with multiple copies, while for GFP and 35S promoter were 70%-90%, in which symmetrical methylation of CG and CNG reached 90%. Cytosines methylation degree of nptⅡin multiple copies transgenic Bingtangcheng line was 10%-40%, GFP and 35S promoter in these lines were 20%-60% and 60%-80%, respectively.
5. Expression of exogenous genes in transgenic citrus. Expression of GFP and nptⅡanalysis by Northern blot and Real-time RT-PCR showed no difference on nptⅡexpression among transgenic plants with different copy transgene numbers. GFP was low expressed in most of the multiple copies transgenic plants. Especially, GFP showed extremely low transcriptional level in transgene silenced plants. There was no correlation between the expression of transgene and copy number in LFY transgenic GWZ or Bingtangcheng.
6 Real-time PCR was applied to screen transgenic Ponkan with single copy. The result indicated that 11 of 43 AP1 transgenic Ponkan had single copy, consistent with the result of Southern blot. The correlation coefficient between results of Real-time PCR and Southern blot of API, GUS and nptⅡwas 0.7521,0.5856 and 0.8222, respectively.
7 Flowering of AP1 or LFY transgenic citrusplants also were investigated. One of the five AP1 transgenic 'Meiwa' kumquat started flowering 11 months after transfer to greenhouse (Duan,2006) and continued to flower in the next two years, other two transgenic plants and three of seven non-transformed plants flowered after 23 months and continued to flower in the next year, In the third year, one transgenic plants delayed flowering when other transgenic or no transgenic plants all flowered. Two of the three LFY transgenic GWZ started flowering one year after transfer to greenhouse. One of the eight LFY transgenic Bingtangcheng was early flowering after transfer to greenhouse two years. Other transgenic or non-transgenic plants did not flower.
8 Expression of exogenous AP1, LFY and endogenous flowering related genes. The expression of exogenous AP1 was different among AP1 transgenic 'Meiwa' kumquat which flowered. CiLFY was up-regulated in some of the flowering transgenic plants, while CiAP1 was down-regulated. In LFY transgenic GWZ and Bingtangcheng, the expression of exogenous LFY was higher in early flowering transgenic plants. Endogenous CiAPl up-regulation and CiLFY down-regulation were found in most LFY transgenic plants. Endogenous CiLFY and CiFT up-regulation, CiTFL1 down-regulation may be related to early flowering of AP1 transgenic 'Meiwa' kumquat. CiAP1 and CiTFL1 up-regulation, CiFT down-regulation may be assosiated with delay flowering of AP1 transgenic 'Meiwa' kumquat. CiFT, CiWUS and CiTFL1 were down-regulated in all early flowering LFY transgenic GWZ and Bingtangcheng. The results indicated that down-regulated of CiTFL1 play a decisive role in flowering time regulation of transgenic AP1 or LFY citrus.
In this study, impact on transgene expression by exogenous gene copy number, T-DNA repeat, methylation of tansgene and its promoter was explored. Additionaly, feasibility of Real-time PCR on gene copy number analysis, the interaction of AP1 or LFY and endogenous flowering genes in controlling juvenile phase were discussed.
引文
1.程运江.柑橘体细胞胞质遗传及叶绿体SSR引物开发研究.[博士学位论文].武汉：华中农业大学图书馆,2004
2.段艳欣.根癌农杆菌介导的柑橘转化体系优化与转LFY, API基因植株的培育.[博士学位论文].武汉：华中农业大学图书馆,2006
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