芒果SCoT分子标记与逆境和重要开花时间相关基因研究
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摘要
芒果是重要的热带水果之一,有“热带水果之王”的美誉。我国芒果种植历史悠久,种质资源丰富且品种繁多,其中许多芒果品种来源于实生选种,亲缘关系不明。长期以来我国芒果主栽品种、国外引进芒果品种以及地方特有芒果品种之间的遗传多样性和亲缘关系还不够清楚,给芒果种质资源的收集、保护和利用带来了诸多不利。近年来由于全球气候变化,导致非生物胁迫经常发生,而芒果对非生物胁迫比较敏感,非生物胁迫严重影响芒果的品质与产量。在提高芒果产量的同时又要保障经济效益,对芒果进行产期调节是增加芒果经济效益的有效途径。四季芒成花不需要低温,具有周年自然成花的特性,是目前进行芒果产期调节的最适宜品种。而关于四季芒成花的分子机理还未见报道。基于上述原因,本研究将率先采用新型目标性状SCoT分子标记对芒果进行遗传多样性和亲缘关系分析,改良cDNA-SCoT技术并首次应用于芒果逆境胁迫差异显示研究,克隆四季芒重要开花时间基因,并对其生物信息学、表达模式和功能进行研究。主要研究结果如下：
1、首次运用SCoT分子标记对芒果遗传多样性进行研究。开发了44条新的SCoT引物,使SCoT分子标记引物总数达到80条。开展了芒果SCoT分子标记和ISSR分子标记比较研究,结果显示SCoT分子标记扩增多态性百分比高于ISSR分子标记,SCoT分子标记的聚类结果比ISSR分子标记更能准确反映研究样品之间的亲缘关系,不同地域来源的芒果品种能按照其起源聚类。证明SCoT分子标记在芒果遗传多样性和亲缘关系研究中是一种非常有效的分子标记。
2、改良cDNA-SCoT技术并首次应用于芒果逆境胁迫差异显示研究。在cDNA的3’末端加锚定引物,利用SCoT分子标记引物结合3’末端锚定引物对cDNA进行扩增,可以保证扩增片段是功能基因,改良的cDNA-SCoT技术方法操作简单、实验成本低廉且节省时间。用37条SCoT分子标记引物结合3’末端加尾锚定引物对逆境胁迫处理和对照芒果样品进行差异显示研究,成功获得92个基因片段,其中与逆境胁迫、转录因子、生长发育和信号传导相关的基因共有40个。
3、改良5’末端RACE技术克隆芒果逆境相关基因的全长。采用新的逆转录引物,增加cDNA产物和接头引物浓度,延长加尾时间,简化操作步骤,结合巢式PCR和降落PCR获得了一种操作简单、快速、高效、成本低廉的改良5’末端RACE方法。利用改良的5’末端RACE技术对cDNA-SCoT获得的芒果逆境相关基因的5’末端进行调取,成功获得40个芒果逆境相关基因的全长序列。
4、芒果逆境胁迫相关基因表达模式分析。从40个逆境相关基因中选取6个逆境功能基因、6个转录因子和4个保护酶基因,利用实时荧光定量技术进行表达模式分析,低温、NaCl和PEG处理均能调控这些基因的表达,不同基因在同一逆境胁迫下的表达模式存在差异,在这三类基因中,转录因子对逆境胁迫的响应最明显。
5、芒果重要逆境基因Harpin基因研究。利用cDNA-SCoT技术和5’末端RACE技术获得芒果Harpin基因全长。Harpin基因全长为1019bp,开放阅读框长666bp,编码222个氨基酸,与其它病原菌的hrp基因无同源性。芒果Harpin基因在各个组织中均表达,并随着果实发育,其表达水平持续上升；低温、NaCl、PEG、铅、镉和锌可诱导该基因表达；果实采后不同处理也诱导该基因表达。芒果Harpin基因转化烟草,其T0代植株高度比对照高,开花时间早于对照,并引起花形态发生变异；用NaCl、Pb(NO3)2和ZnSO4处理转Harpin基因烟草,T0代植株表型与对照差异不明显。芒果Harpin基因功能还需要进一步对T1和T2转基因植株进行研究才能确定。
6、利用同源克隆法、染色体步移法或改良5'RACE法,从芒果中获得了LFY,CO、SOC1、FT和TFL1共5个开花时间基因的序列全长,并对这些基因的生物信息学、表达模式及其功能进行研究。
基因生物信息学分析：LFY基因基因全长为1332bp,其中开放阅读框长1149bp,编码383个氨基酸,存在一个FLO_LFY superfamily结构域；LFY基因的启动子区域包含多个逆境和光响应元件,同时还存在ABA、GA、CTK、IAA和蔗糖响应元件。CO基因开放阅读框长为966bp,编码322个氨基酸,存在两个B-box锌子结构域和一个CCT结构域,属于第一类CO基因家族,与拟南芥COL4基因最相似。SOC1基因开放阅读框长为669bp,编码223个氨基酸,存在一个MADS结构域和一个K-box结构域,属于MADS-box基因家族,不同物种的SOC1基因编码氨基酸数差异很小FT基因开放阅读框长为588bp,编码196个氨基酸,存在一个PEBP结构域,属于FT/TFL1基因家族的FT-like类基因,但其编码氨基酸数明显多于其它FT/TFL1基因。TFL1基因开放阅读框长为516bp,编码172个氨基酸,也存在一个PEBP结构域,属于第一类FT,TFL1基因家族的TFL1-like类基因。
基因表达模式分析：LFY和CO基因在营养组织和生殖组织中均表达,而SOCl基因主要在营养组织中表达。LFY、CO、SOC1、FT和TFL1基因在紫花芒和四季芒的成熟叶、成熟茎和嫩茎中的表达模式既类似又存在明显差异,在芒果花芽分化期的10月-12月份之间这些基因都具有较高的表达水平,在来年的3月-6月份之间还存在另外一个小的表达高峰；但在同一时间不同组织、同一组织不同时间以及不同芒果品种之间表达高峰出现的时间以及表达水平都存在差异；进一步分析显示,芒果开花时间基因之间也存在网络调控。
基因功能研究：分别构建了LFY、CO、SOC1、FT和TFL1基因的正义和反义表达载体,并转化烟草。不同转基因烟草插入拷贝数存在差异,并且芒果基因在转基因烟草中正常表达。分别转LFY+、CO+、FT-和TFL1-基因的烟草,转基因T0代株高低于对照的株高,而分别转LFY-、CO-、SOC-、FT+和TFLl+基因的烟草,其T0代株高比对照烟草的株高高,SOC+转基因株高与对照差异不明显。在本研究中,这些芒果开花时间基因转化烟草后,转基因引起烟草花形态发生变异,但T0代转基因烟草开花时间未能达到预想效果,其功能还需要进一步验证。
Mango(Mangifera indica L) is one of the important tropical fruit, known as the king of fruits.There are a long history of cultivation, rich germplasm resources and many varieties or cultivars about mango in China. Because many mango cultivars were selected from naturally occurring open-pollinated seedlings, their parents were unknown. The genetic relationships among main cultivars, cultivars introduced and some local unique cultivars of mango in our country were unclear, which brought a lot of problems for mango germplasm resources collection, protection and utilization. Abiotic stresses often occur by the global climate change, while mango is sensitive to abiotic stress, therefore the abiotic stresses are major environmental factors that influence the quality and yield of mango seriously. Off-season of mango is the right chose to increase yield and benefit at the same time. Mangifera indica L. cv.'Chok Anand' can blossom several times without low temperature of the year, which is the most suitable for off-season production, while there have been no reports on the molecular mechanism of flowering about Mangifera indica L. cv.'Chok Anand'. Based on the above reasons, SCoT marker technique was firstly used to study the genetic diversity and relationships among mango accessions, cDNA-SCoT technology improved was first applied to isolate and characterize differential genes expressed under stress treated, and full length cloning, bioinformatics, expression patterns and preliminary functions of important flowering time genes were studied in the present study. The main results were as follows:
1. In the present study, SCoT markers were first applied to identify the genetic diversity and relationships among mango cultivars.44new SCoT primers were designed, and the total number of SCoT primers amount to80. The usefulness of SCoT versus ISSR markers for identiying the closest mango cultivars was evaluated, the polymorphic percentages amplified by SCoT markers were higher than that of ISSR markers, and the UPGMA cluster result with SCoT markers was better than that with ISSR analysis in their genetic relationships. and mango varieties of the same or near to origin place.could be clustered to the same group by UPGMA cluster with SCoT markers The results demonstrated that the SCoT marker system is very useful in studying genetic diversity and relationship of mango cultivars.
2. The cDNA-SCoT technology was improved and applied in differential gene expression analysis of mango under stresses for the first time. The3'end anchor primer was added to cDNA and combined with SCoT primer for PCR amplification. The cDNA-SCoT technology improved is a simple, low-cost, time saving and efficient method for differential gene expression research, and the fragment amplified by cDNA-SCoT is a function gene.37SCoT primers combined with anchor primer were applied to differential genes expression analysis of mango under stresses in the present study,92fragments were successful obtained and most of these genes were first cloned from mango. The40genes among the92fragments were presumed to be transcription factors and the genes of,stress-related protein, growth development and signal transduction.
3. The5'end RACE technology was modified and applied in cloning the full length of stress related genes in mango. The main modifications for normal5'end RACE were as followed:Using new reverse transcriptase primer, increasing the concentration of cDNA and tail primer, extending tailing time, simplifying the process, combined with nested PCR and touchdown PCR. The5' end RACE technology modified was testified to be a new, simple, rapid, high-efficient and low-cost method. In the present study,40full-length genes related with stress were successful obtained by using the51end RACE technology modified.
4. Expression analysis of the important stress-related genes in mango. Expression patterns of six stress-responsive functional genes, six transcription factors and four protective enzyme genes were estimated by real-time quantitative PCR, the results showed that these genes were all regulated by low temperture, NaCl and PEG treatments, but there were different expression patterns under the same stress treatment. The transcription factors among the three kinds of stress-related genes were strong response to the stresses.
5. Cloning, expression and function analysis Harpin gene of mango. The full length of mango Harpin gene was obtained by cDNA-SCoT technology and RACE technology. The full length of Harpin cDNA is1019bp and contains a complete open reading frame (ORF) of666bp, which encoding222amino acids. The sequence of mango Harpin is no homology with pathogenic bacteria hrp genes. Harpin gene of mango can express in all tissues, and the expression level rise continuely with the fruit development. The expression analysis revealed mango Harpin gene was regulated by low temperature, NaCl, PEG, heavy metal and different treatments after fruit harvest. Sense Harpin vector of mango was successful transformed into tobacco. Sense transgenic plants leaded to early flowering, increase plant height and cause flower structure mutant. More treatments including NaCl, Pb(NO3)2and ZnSO4all did not differ significantly in To generations between transgenic plants and non-transgenic plants. The function of mango Harpin gene could be identified by further studying the T1and T2transgenic plants.
6. The full length of mango flowering time genes LFY, CO, SOC1, FT and TFL1were obtained by homology cloning strategy, genome walking and5'end RACE technology modified, and their bio informatics, expression patterns and functions of these genes have been further studied.
Bio informatics analysis:The full length of LFY cDNA is1332bp and contains a complete open reading frame (ORF) of1149bp, encoding383amino acids. The LFY protein contains one conserved FLO_LFY superfamily domains. LFY promoter contains several cis-regulatory elements related to stress-induce and light-response, and cis-acting elements involved in ABA-, GA-, CTK-, IAA-and sugar-responsive were also identified in the promoter. The full length of CO cDNA is1141bp and contains a complete open reading frame (ORF) of966bp, encoding322amino acids. The CO protein contains two B-box domains and one CCT domains, belong to the first group of CO gene, and is close to Arabidopsis AtCOL4gene. The full length of SOC1cDNA is841bp and contains a complete open reading frame (ORF) of669bp, encoding223amino acids. The SOC1protein contains one MADS-box domains and one K-box domains, belong to MADS-box gene family. The full length of FT cDNA is781bp and contains a complete open reading frame (ORF) of588bp, encoding196amino acids. The FT protein contains one PEBP domains, belong to FT/TFL1gene family. The full length of TFL1cDNA is721bp and contains a complete open reading frame (ORF) of516bp, encoding172amino acids. The TFL1protein contains one PEBP domains, belong to FT/TFL1gene family.
Expression pattern analysis:Real-time-PCR analysis showed that LFY and CO expressed in all vegetative tissues and reproductive organs, but SOC1gene mainly expressed in vegetative tissues. The expression patterns of LFY, CO, SOC1, FT and TFL1in mature leaves, mature stems and young stems in two cultivars of Mangifera indica L. cv.'Chok Anand'and Mangifera indica L. cv.'ZiHua'were almost similar, but also exist obvious difference. The flowering time genes expressed highly during floral induction from November to December, and another peak appeared from March to June in the following year. Whereas the time of peak appeared and the expression level of these genes were different in different tissues at the same time, in the same tissues at different time and in different mango varieties, further analysis showed that mango flowering time genes also existed regulation network.
Function analysis:The sense and antisense vector of the five mango flowering time genes were constructed and successful transferred into tobacco, respectively. The copy numbers were differences between transgenic plants, and these flowering time genes normally expressed in the transgenic plants. The plant heights of the To generations of transgenic plants with LFY+、CO+、FT-and TFL-respectively were lower than that of non transgenic plants, but the plant heights of the To generations of transgenic plants with LFY-、CO-、SOC-、FT+and TFL+respectively showed higher. The SOC+transgenic plants did not differ significantly between transgenic plants and non transgenic plants. In the present study, floral organs of transgenic tobacco To generations with mango flowering time genes had happened mutants, but flowering time have not worked as well as had been hoped,. The functions of these genes could be further studied by the T1and T2transgenic plants.
1、首次运用SCoT分子标记对芒果遗传多样性进行研究。开发了44条新的SCoT引物,使SCoT分子标记引物总数达到80条。开展了芒果SCoT分子标记和ISSR分子标记比较研究,结果显示SCoT分子标记扩增多态性百分比高于ISSR分子标记,SCoT分子标记的聚类结果比ISSR分子标记更能准确反映研究样品之间的亲缘关系,不同地域来源的芒果品种能按照其起源聚类。证明SCoT分子标记在芒果遗传多样性和亲缘关系研究中是一种非常有效的分子标记。
2、改良cDNA-SCoT技术并首次应用于芒果逆境胁迫差异显示研究。在cDNA的3’末端加锚定引物,利用SCoT分子标记引物结合3’末端锚定引物对cDNA进行扩增,可以保证扩增片段是功能基因,改良的cDNA-SCoT技术方法操作简单、实验成本低廉且节省时间。用37条SCoT分子标记引物结合3’末端加尾锚定引物对逆境胁迫处理和对照芒果样品进行差异显示研究,成功获得92个基因片段,其中与逆境胁迫、转录因子、生长发育和信号传导相关的基因共有40个。
3、改良5’末端RACE技术克隆芒果逆境相关基因的全长。采用新的逆转录引物,增加cDNA产物和接头引物浓度,延长加尾时间,简化操作步骤,结合巢式PCR和降落PCR获得了一种操作简单、快速、高效、成本低廉的改良5’末端RACE方法。利用改良的5’末端RACE技术对cDNA-SCoT获得的芒果逆境相关基因的5’末端进行调取,成功获得40个芒果逆境相关基因的全长序列。
4、芒果逆境胁迫相关基因表达模式分析。从40个逆境相关基因中选取6个逆境功能基因、6个转录因子和4个保护酶基因,利用实时荧光定量技术进行表达模式分析,低温、NaCl和PEG处理均能调控这些基因的表达,不同基因在同一逆境胁迫下的表达模式存在差异,在这三类基因中,转录因子对逆境胁迫的响应最明显。
5、芒果重要逆境基因Harpin基因研究。利用cDNA-SCoT技术和5’末端RACE技术获得芒果Harpin基因全长。Harpin基因全长为1019bp,开放阅读框长666bp,编码222个氨基酸,与其它病原菌的hrp基因无同源性。芒果Harpin基因在各个组织中均表达,并随着果实发育,其表达水平持续上升；低温、NaCl、PEG、铅、镉和锌可诱导该基因表达；果实采后不同处理也诱导该基因表达。芒果Harpin基因转化烟草,其T0代植株高度比对照高,开花时间早于对照,并引起花形态发生变异；用NaCl、Pb(NO3)2和ZnSO4处理转Harpin基因烟草,T0代植株表型与对照差异不明显。芒果Harpin基因功能还需要进一步对T1和T2转基因植株进行研究才能确定。
6、利用同源克隆法、染色体步移法或改良5'RACE法,从芒果中获得了LFY,CO、SOC1、FT和TFL1共5个开花时间基因的序列全长,并对这些基因的生物信息学、表达模式及其功能进行研究。
基因生物信息学分析：LFY基因基因全长为1332bp,其中开放阅读框长1149bp,编码383个氨基酸,存在一个FLO_LFY superfamily结构域；LFY基因的启动子区域包含多个逆境和光响应元件,同时还存在ABA、GA、CTK、IAA和蔗糖响应元件。CO基因开放阅读框长为966bp,编码322个氨基酸,存在两个B-box锌子结构域和一个CCT结构域,属于第一类CO基因家族,与拟南芥COL4基因最相似。SOC1基因开放阅读框长为669bp,编码223个氨基酸,存在一个MADS结构域和一个K-box结构域,属于MADS-box基因家族,不同物种的SOC1基因编码氨基酸数差异很小FT基因开放阅读框长为588bp,编码196个氨基酸,存在一个PEBP结构域,属于FT/TFL1基因家族的FT-like类基因,但其编码氨基酸数明显多于其它FT/TFL1基因。TFL1基因开放阅读框长为516bp,编码172个氨基酸,也存在一个PEBP结构域,属于第一类FT,TFL1基因家族的TFL1-like类基因。
基因表达模式分析：LFY和CO基因在营养组织和生殖组织中均表达,而SOCl基因主要在营养组织中表达。LFY、CO、SOC1、FT和TFL1基因在紫花芒和四季芒的成熟叶、成熟茎和嫩茎中的表达模式既类似又存在明显差异,在芒果花芽分化期的10月-12月份之间这些基因都具有较高的表达水平,在来年的3月-6月份之间还存在另外一个小的表达高峰；但在同一时间不同组织、同一组织不同时间以及不同芒果品种之间表达高峰出现的时间以及表达水平都存在差异；进一步分析显示,芒果开花时间基因之间也存在网络调控。
基因功能研究：分别构建了LFY、CO、SOC1、FT和TFL1基因的正义和反义表达载体,并转化烟草。不同转基因烟草插入拷贝数存在差异,并且芒果基因在转基因烟草中正常表达。分别转LFY+、CO+、FT-和TFL1-基因的烟草,转基因T0代株高低于对照的株高,而分别转LFY-、CO-、SOC-、FT+和TFLl+基因的烟草,其T0代株高比对照烟草的株高高,SOC+转基因株高与对照差异不明显。在本研究中,这些芒果开花时间基因转化烟草后,转基因引起烟草花形态发生变异,但T0代转基因烟草开花时间未能达到预想效果,其功能还需要进一步验证。
Mango(Mangifera indica L) is one of the important tropical fruit, known as the king of fruits.There are a long history of cultivation, rich germplasm resources and many varieties or cultivars about mango in China. Because many mango cultivars were selected from naturally occurring open-pollinated seedlings, their parents were unknown. The genetic relationships among main cultivars, cultivars introduced and some local unique cultivars of mango in our country were unclear, which brought a lot of problems for mango germplasm resources collection, protection and utilization. Abiotic stresses often occur by the global climate change, while mango is sensitive to abiotic stress, therefore the abiotic stresses are major environmental factors that influence the quality and yield of mango seriously. Off-season of mango is the right chose to increase yield and benefit at the same time. Mangifera indica L. cv.'Chok Anand' can blossom several times without low temperature of the year, which is the most suitable for off-season production, while there have been no reports on the molecular mechanism of flowering about Mangifera indica L. cv.'Chok Anand'. Based on the above reasons, SCoT marker technique was firstly used to study the genetic diversity and relationships among mango accessions, cDNA-SCoT technology improved was first applied to isolate and characterize differential genes expressed under stress treated, and full length cloning, bioinformatics, expression patterns and preliminary functions of important flowering time genes were studied in the present study. The main results were as follows:
1. In the present study, SCoT markers were first applied to identify the genetic diversity and relationships among mango cultivars.44new SCoT primers were designed, and the total number of SCoT primers amount to80. The usefulness of SCoT versus ISSR markers for identiying the closest mango cultivars was evaluated, the polymorphic percentages amplified by SCoT markers were higher than that of ISSR markers, and the UPGMA cluster result with SCoT markers was better than that with ISSR analysis in their genetic relationships. and mango varieties of the same or near to origin place.could be clustered to the same group by UPGMA cluster with SCoT markers The results demonstrated that the SCoT marker system is very useful in studying genetic diversity and relationship of mango cultivars.
2. The cDNA-SCoT technology was improved and applied in differential gene expression analysis of mango under stresses for the first time. The3'end anchor primer was added to cDNA and combined with SCoT primer for PCR amplification. The cDNA-SCoT technology improved is a simple, low-cost, time saving and efficient method for differential gene expression research, and the fragment amplified by cDNA-SCoT is a function gene.37SCoT primers combined with anchor primer were applied to differential genes expression analysis of mango under stresses in the present study,92fragments were successful obtained and most of these genes were first cloned from mango. The40genes among the92fragments were presumed to be transcription factors and the genes of,stress-related protein, growth development and signal transduction.
3. The5'end RACE technology was modified and applied in cloning the full length of stress related genes in mango. The main modifications for normal5'end RACE were as followed:Using new reverse transcriptase primer, increasing the concentration of cDNA and tail primer, extending tailing time, simplifying the process, combined with nested PCR and touchdown PCR. The5' end RACE technology modified was testified to be a new, simple, rapid, high-efficient and low-cost method. In the present study,40full-length genes related with stress were successful obtained by using the51end RACE technology modified.
4. Expression analysis of the important stress-related genes in mango. Expression patterns of six stress-responsive functional genes, six transcription factors and four protective enzyme genes were estimated by real-time quantitative PCR, the results showed that these genes were all regulated by low temperture, NaCl and PEG treatments, but there were different expression patterns under the same stress treatment. The transcription factors among the three kinds of stress-related genes were strong response to the stresses.
5. Cloning, expression and function analysis Harpin gene of mango. The full length of mango Harpin gene was obtained by cDNA-SCoT technology and RACE technology. The full length of Harpin cDNA is1019bp and contains a complete open reading frame (ORF) of666bp, which encoding222amino acids. The sequence of mango Harpin is no homology with pathogenic bacteria hrp genes. Harpin gene of mango can express in all tissues, and the expression level rise continuely with the fruit development. The expression analysis revealed mango Harpin gene was regulated by low temperature, NaCl, PEG, heavy metal and different treatments after fruit harvest. Sense Harpin vector of mango was successful transformed into tobacco. Sense transgenic plants leaded to early flowering, increase plant height and cause flower structure mutant. More treatments including NaCl, Pb(NO3)2and ZnSO4all did not differ significantly in To generations between transgenic plants and non-transgenic plants. The function of mango Harpin gene could be identified by further studying the T1and T2transgenic plants.
6. The full length of mango flowering time genes LFY, CO, SOC1, FT and TFL1were obtained by homology cloning strategy, genome walking and5'end RACE technology modified, and their bio informatics, expression patterns and functions of these genes have been further studied.
Bio informatics analysis:The full length of LFY cDNA is1332bp and contains a complete open reading frame (ORF) of1149bp, encoding383amino acids. The LFY protein contains one conserved FLO_LFY superfamily domains. LFY promoter contains several cis-regulatory elements related to stress-induce and light-response, and cis-acting elements involved in ABA-, GA-, CTK-, IAA-and sugar-responsive were also identified in the promoter. The full length of CO cDNA is1141bp and contains a complete open reading frame (ORF) of966bp, encoding322amino acids. The CO protein contains two B-box domains and one CCT domains, belong to the first group of CO gene, and is close to Arabidopsis AtCOL4gene. The full length of SOC1cDNA is841bp and contains a complete open reading frame (ORF) of669bp, encoding223amino acids. The SOC1protein contains one MADS-box domains and one K-box domains, belong to MADS-box gene family. The full length of FT cDNA is781bp and contains a complete open reading frame (ORF) of588bp, encoding196amino acids. The FT protein contains one PEBP domains, belong to FT/TFL1gene family. The full length of TFL1cDNA is721bp and contains a complete open reading frame (ORF) of516bp, encoding172amino acids. The TFL1protein contains one PEBP domains, belong to FT/TFL1gene family.
Expression pattern analysis:Real-time-PCR analysis showed that LFY and CO expressed in all vegetative tissues and reproductive organs, but SOC1gene mainly expressed in vegetative tissues. The expression patterns of LFY, CO, SOC1, FT and TFL1in mature leaves, mature stems and young stems in two cultivars of Mangifera indica L. cv.'Chok Anand'and Mangifera indica L. cv.'ZiHua'were almost similar, but also exist obvious difference. The flowering time genes expressed highly during floral induction from November to December, and another peak appeared from March to June in the following year. Whereas the time of peak appeared and the expression level of these genes were different in different tissues at the same time, in the same tissues at different time and in different mango varieties, further analysis showed that mango flowering time genes also existed regulation network.
Function analysis:The sense and antisense vector of the five mango flowering time genes were constructed and successful transferred into tobacco, respectively. The copy numbers were differences between transgenic plants, and these flowering time genes normally expressed in the transgenic plants. The plant heights of the To generations of transgenic plants with LFY+、CO+、FT-and TFL-respectively were lower than that of non transgenic plants, but the plant heights of the To generations of transgenic plants with LFY-、CO-、SOC-、FT+and TFL+respectively showed higher. The SOC+transgenic plants did not differ significantly between transgenic plants and non transgenic plants. In the present study, floral organs of transgenic tobacco To generations with mango flowering time genes had happened mutants, but flowering time have not worked as well as had been hoped,. The functions of these genes could be further studied by the T1and T2transgenic plants.
引文
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