苎麻性别决定及相关基因的基础研究
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摘要
苎麻是重要的纤维作物，且苎麻是多年生无性繁殖为主的异花授粉作物，杂种优势明显，杂种优势利用潜力较大。由于苎麻品种高度杂合性，苎麻杂种F_1代性状严重分离；另外由于苎麻是雌雄同株作物，杂种优势利用中需要隔离和去雄，费时费力；这是长期困扰苎麻杂种优势利用的两大难题。再者，由于苎麻的杂合性，种子繁殖会造成后代的严重退化，生产中常用无性繁殖方法，但是无性繁殖成本高，速度慢，且易带病虫，这也是苎麻生产中遇到的问题。苎麻的性别决定是苎麻杂交优势利用及苎麻繁殖方法改进的基础。
关于苎麻性别决定的研究，前人观察表明苎麻雄花序分化过程中先形成雌雄蕊原始体，然后雌蕊退化形成雄花。而雌花中没有发现雄蕊痕迹。但是并没有给出雌雄蕊决定的准确时间，也没有外部形态标记。GA_3诱导苎麻属全雌性无融合生殖种产生雄花的试验表明，花芽分化至雌蕊原基分化形成之前(花芽长度0.5cm左右)比较容易诱导性转换。但是此试验并没有给出性别决定的确切时间点和外部形态特征标记。外源激素与苎麻性别的关系的研究方面主要集中在性别逆转上。关于苎麻性别决定相关基因方面的研究迄今为止未见报道。
本研究利用性别表达特异的材料，对其植物学性状、生长动态、花芽分化、内源乙烯、苎麻乙烯合成关键酶(ACC合成酶)基因的克隆及时空表达特性等几个方面进行研究，结果如下：
1)对性别表现特异的雌株苎麻(GBN-09)和雌雄同株苎麻(GBN-08)进行田间调查，发现GBN-08叶柄微红、托叶合生，茎黄绿色，花蕾绿色，茎斑细小且稀疏，而GBN-09叶柄绿色、托叶分生，茎绿色，花蕾黄白，茎斑大且密集。
2)对雌株苎麻(GBN-09)和雌雄同株苎麻(GBN-08)进行生长动态调查，发现GBN-08和GBN-09二麻期的株高日生长量呈抛物线，三麻期的株高日生长量逐渐降低；在二麻和三麻期，节位数的日增加量以前期增加较快，后期增加减缓为主；叶长与叶宽生长动态与株高的生长动态相同，而且叶片的伸长与加宽是正相关的。
3)对雌株苎麻(GBN-09)和雌雄同株苎麻(GBN-08)进行开花特性调查，发现苎麻第一花芽(最先分化的花芽)的性别决定整株的性别，即如第一花芽是雌性，则整株是雌株；如第一花芽是雄性则整株是雌雄同株。
4)对GBN-08和GBN-09的花芽分化进行显微切片观察，发现第一花芽大小为0.20±0.067cm是雌雄同株苎麻GBN-08的性别决定期，此时总节位为28-30节；第一花芽大小为0.25±0.037cm时是雌株苎麻GBN-09的性别决定期，此时总节位为24-29节。
5)对不同性别苎麻茎尖乙烯释放速率、不同性别苎麻材料不同节位花芽乙烯释放速率、雌雄同株苎麻的雌花序、雄花序和雌雄混合花序的乙烯释放速率进行测定，同时对外施乙烯抑制剂(AVG和AgNO_3)对苎麻性别的控制进行研究，表明苎麻性别分化与内源乙烯密切相关，高水平乙烯释放速率诱导雌株苎麻或雌花的产生。雌株苎麻在茎尖、叶和花芽中的乙烯含量都大于雌雄同株苎麻。在雌雄同株苎麻中，乙烯在雌花序中的含量高于在混合花序和雄花序中的含量。乙烯抑制剂AVG和AgNO_3可以控制苎麻的性别表现，以乙烯合成抑制剂AVG效果好，且以300mg／L为最佳诱导剂浓度。
6)通过比较几种RNA提取试剂对雌花，果实，叶片，茎皮的总RNA提取效果，发现RNAplant可以有效地提取苎麻不同组织的RNA且质量很好，可以进行mRNA的抽提，cDNA的合成，RT-PCR等下游实验。
7)根据已克隆的双子叶植物的ACC合成酶基因序列设计简并引物，以苎麻茎尖总RNA为模板，通过RT-PCR结合NEST-PCR扩增得到615bp的cDNA序列。对得到的cDNA序列在NCBI网站进行BLASTn和BLASTx比对显示此序列是苎麻ACC合成酶基因，编码205个氨基酸。苎麻ACC合成酶基因推导的氨基酸与苹果、拟南芥、大豆、番茄、甘蓝、黄瓜、柑桔、烟草、桃的蛋白结构域的保守性分析显示含有以报道的ACC合成酶基因5个保守区。同源树分析表明，苎麻ACC合成酶基因与大豆ACC合成酶基因的同源性最高。
8)实时定量PCR分析苎麻ACC合成酶基因的时空表达，发现在苗期以茎尖的表达量较高，叶片和茎杆次之，根中最低。在开花期和结果期，该基因的表达都比较低。当第一花芽大小为0.75cm时(处于花芽分化后期至开花前期之间)，雌株苎麻(GBN-09)的茎尖ACC合成酶的表达量是苗期茎尖的26.2倍，雌雄同株苎麻(GBN-08)的茎尖表达量是苗期茎尖的10.1倍；而对于花芽来说，ACC合成酶基因表达量同样以雌株苎麻高于雌雄同株苎麻，它们分别是苗期茎尖的9.36和1.27倍。
综上所述，本研究在植物学性状，花芽分化，内源乙烯、乙烯合成关键酶(ACC合成酶)基因以及苎麻ACC合成酶基因时空表达特点与苎麻性别的关系等方面取得了显著的新进展，对苎麻性别控制、杂种优势利用和改进苎麻繁殖方法等方面具有重要的理论和实践价值。
Rarnie(Boehmeria nivea (L.) Gaud.) is perennial bast fiber crop. Scince ramie is a cross-pollinatedcultivated plant with complex genetic background, its offspring can be badly segregated. It was moreexpensive that the male flower must be deleted in heterosis and breeding by clone method. To solvethese problem, it is very important to study on sexual determination and related genes to control ramiesexual expression by researching bud morphology and differentiation, physiology and moleculargenetics in sexual special ramie. This research could promote to ulitize the heterosis and improve on thebreeding method.
About the studis on sexual determination of ramie, the gynandrous primordium was formed firstlyduring sex differentiation in male flower. Then the male flower could be shaped by degenerated thegynoecium. But the androecium was not appeared in female flower. When the bud growed to 0.5centimeter before the the gynandrous primordium was formed, the male were easily induced by GA_3among apomixes species. The exact time and maker of external morphology that happened at sexualdetermination date in ramie were not known.The study of relation between the exogenous hormone andsex was mostly focus on reversing sex in ramie.It was not reported about the related genes of sexdetermination in ramie.
In this paper, the ramie materials with unusual sex was studied on observing in botany character,researching bud differentiation, mensurating endogenesis ethylene content, spraying the ethyleneinhibitor, cloning the ACC synthase and studying spatio-temporal expression.The main result of thispaper are as follows:
1. The female ramie (GBN-09) and hermaphrodite ramie(GBN-08) were investigated in second crop.
There are light red leafstalk, connate stipul, kelly stem, green bud and small stem spot in GBN-08.it was different with GBN-09 that have green leafstalk, asunder stipul, white yellow bud and bigstem spot.
2. The growth trend was obtained by observing the GBN-09 and GBN-O8.The result showed the daygrowth trends of stem highness was parabola on GBN-08 and GBN-09 in the second crops, wasgradually reduced in third crops. The day growth trends of node numebers was faster ininitiat-stage than late-stage. It is the same growth trends between the stem highness and leaf lengthand leaf width, and the growth trends is positive correlation between the leaf length and leaf width.
3. The characteristic of flowering were studied in female ramie (GBN-09) and hermaphrodite ramie(GBN-08). The result showed that the sex of individual plant of ramie was determined by the fastbud (which was earliest differentiation). If the fast bud was female, that the individual plant oframie was female. But if the the first bud was male, that the individual plant of ramie washermaphrodite.
4. Morphological observation of flower bud differentiation were done with GBN-08 and GBN-09 byparaffin section. It was the time of sex determination in hermaphrodite ramie including 28-30 node When the bud was 0.2±0.067 centimeter; And It was the time of sex determination in female ramieincluding 24-29 node When the bud was 0.25±0.037 centimeter.
5. The ethylene release rate was measured in shoot tip of different sex ramie in different time, the budof same node in female and hermaphrodite ramie in second crops and different sex singleinflorescence in hermaphrodite ramie. The result indicated there was closed relationship betweensexual different ramie and ethylene release rate. The female ramie could be induced by highethylene release rate. the ethylene content in ramie plant with female flower is higher than ramieplant with androgynary in stem apex, leaf and flower. In flower organ, the ethylene content ishigher in female flower than male flower and mixed inflorescence. The sex expression of ramiewas found to be controlled by foliar spray with 2-aminoethoxyvinylglycine(AVG) and AgNO3.AVG at a concentration of 300,100mg/L and AgNO3 at a 100, 300,500mg/L caused increase ofmaleness and inhibition of fe-male flower. AVG at a concentration of 300mg/L were the best.
6. It is necessary to extract total RNA from plant tissue.A new reagent—RNAplant was screened outto extract RNA from ramie leaf, stern, female flower and fruit by used five different reagents.RNAswere effective extracted and the high quality RNA can be used for cDNA synthesis, RT-PCR andothers downstream experiment.
7. ACC synthase(ACS) was a key enzyme for ethylene biosynthesis. In this paper, four degenerateoligonucleotide primers were designed, coding for consernative amino acid regions in ACCsynthase protein family of dicotyledon. Reverse transcription PCR and Nest PCR were performedon total RNA extracted from shoot tip of ramie, and produced 615bp fragment. By using theprogram of BLASTn and BLASTx on NCBI GenBank database, the fragment was ACC synthasegene, coded 205 amino acids. Domain of deduced ACS amino acids in ramie was aligned withACS amino acids of twenty plant. The result showed that there are five conserve domain in ACSamino acids of ramie.The ramie have higher similar with soybean by phylogeny.
8. To study the differential expression of ACS gene at different development stage and in differenttissues of ramie,relatively quantitative analysis was performed by Realtime-PCR. The expressionlevel of ACS gene in stem apex was higher than leaf, stem and lowest in root in seedling. Theexpression level of ACS gene was lower in anthesis and fruiting period. The expression level ofACS gene in stem apex or bud of GBN-09 was higher than that of GBN-08 when size of the firstbud was 0.75 centimeter.
So, the new progress about the sex determination of ramie was obtained in botany character,researching bud differentiation, mensurating endogenesis ethylene content, spraying the ethyleneinhibitor, cloning the ACC synthase and studying spatio-temporal expression.. The results provided aexprerimental and theoretical basis for controling the sex of ramie, utilizing the hybrid vigor andimproving the breeding methods.
关于苎麻性别决定的研究，前人观察表明苎麻雄花序分化过程中先形成雌雄蕊原始体，然后雌蕊退化形成雄花。而雌花中没有发现雄蕊痕迹。但是并没有给出雌雄蕊决定的准确时间，也没有外部形态标记。GA_3诱导苎麻属全雌性无融合生殖种产生雄花的试验表明，花芽分化至雌蕊原基分化形成之前(花芽长度0.5cm左右)比较容易诱导性转换。但是此试验并没有给出性别决定的确切时间点和外部形态特征标记。外源激素与苎麻性别的关系的研究方面主要集中在性别逆转上。关于苎麻性别决定相关基因方面的研究迄今为止未见报道。
本研究利用性别表达特异的材料，对其植物学性状、生长动态、花芽分化、内源乙烯、苎麻乙烯合成关键酶(ACC合成酶)基因的克隆及时空表达特性等几个方面进行研究，结果如下：
1)对性别表现特异的雌株苎麻(GBN-09)和雌雄同株苎麻(GBN-08)进行田间调查，发现GBN-08叶柄微红、托叶合生，茎黄绿色，花蕾绿色，茎斑细小且稀疏，而GBN-09叶柄绿色、托叶分生，茎绿色，花蕾黄白，茎斑大且密集。
2)对雌株苎麻(GBN-09)和雌雄同株苎麻(GBN-08)进行生长动态调查，发现GBN-08和GBN-09二麻期的株高日生长量呈抛物线，三麻期的株高日生长量逐渐降低；在二麻和三麻期，节位数的日增加量以前期增加较快，后期增加减缓为主；叶长与叶宽生长动态与株高的生长动态相同，而且叶片的伸长与加宽是正相关的。
3)对雌株苎麻(GBN-09)和雌雄同株苎麻(GBN-08)进行开花特性调查，发现苎麻第一花芽(最先分化的花芽)的性别决定整株的性别，即如第一花芽是雌性，则整株是雌株；如第一花芽是雄性则整株是雌雄同株。
4)对GBN-08和GBN-09的花芽分化进行显微切片观察，发现第一花芽大小为0.20±0.067cm是雌雄同株苎麻GBN-08的性别决定期，此时总节位为28-30节；第一花芽大小为0.25±0.037cm时是雌株苎麻GBN-09的性别决定期，此时总节位为24-29节。
5)对不同性别苎麻茎尖乙烯释放速率、不同性别苎麻材料不同节位花芽乙烯释放速率、雌雄同株苎麻的雌花序、雄花序和雌雄混合花序的乙烯释放速率进行测定，同时对外施乙烯抑制剂(AVG和AgNO_3)对苎麻性别的控制进行研究，表明苎麻性别分化与内源乙烯密切相关，高水平乙烯释放速率诱导雌株苎麻或雌花的产生。雌株苎麻在茎尖、叶和花芽中的乙烯含量都大于雌雄同株苎麻。在雌雄同株苎麻中，乙烯在雌花序中的含量高于在混合花序和雄花序中的含量。乙烯抑制剂AVG和AgNO_3可以控制苎麻的性别表现，以乙烯合成抑制剂AVG效果好，且以300mg／L为最佳诱导剂浓度。
6)通过比较几种RNA提取试剂对雌花，果实，叶片，茎皮的总RNA提取效果，发现RNAplant可以有效地提取苎麻不同组织的RNA且质量很好，可以进行mRNA的抽提，cDNA的合成，RT-PCR等下游实验。
7)根据已克隆的双子叶植物的ACC合成酶基因序列设计简并引物，以苎麻茎尖总RNA为模板，通过RT-PCR结合NEST-PCR扩增得到615bp的cDNA序列。对得到的cDNA序列在NCBI网站进行BLASTn和BLASTx比对显示此序列是苎麻ACC合成酶基因，编码205个氨基酸。苎麻ACC合成酶基因推导的氨基酸与苹果、拟南芥、大豆、番茄、甘蓝、黄瓜、柑桔、烟草、桃的蛋白结构域的保守性分析显示含有以报道的ACC合成酶基因5个保守区。同源树分析表明，苎麻ACC合成酶基因与大豆ACC合成酶基因的同源性最高。
8)实时定量PCR分析苎麻ACC合成酶基因的时空表达，发现在苗期以茎尖的表达量较高，叶片和茎杆次之，根中最低。在开花期和结果期，该基因的表达都比较低。当第一花芽大小为0.75cm时(处于花芽分化后期至开花前期之间)，雌株苎麻(GBN-09)的茎尖ACC合成酶的表达量是苗期茎尖的26.2倍，雌雄同株苎麻(GBN-08)的茎尖表达量是苗期茎尖的10.1倍；而对于花芽来说，ACC合成酶基因表达量同样以雌株苎麻高于雌雄同株苎麻，它们分别是苗期茎尖的9.36和1.27倍。
综上所述，本研究在植物学性状，花芽分化，内源乙烯、乙烯合成关键酶(ACC合成酶)基因以及苎麻ACC合成酶基因时空表达特点与苎麻性别的关系等方面取得了显著的新进展，对苎麻性别控制、杂种优势利用和改进苎麻繁殖方法等方面具有重要的理论和实践价值。
Rarnie(Boehmeria nivea (L.) Gaud.) is perennial bast fiber crop. Scince ramie is a cross-pollinatedcultivated plant with complex genetic background, its offspring can be badly segregated. It was moreexpensive that the male flower must be deleted in heterosis and breeding by clone method. To solvethese problem, it is very important to study on sexual determination and related genes to control ramiesexual expression by researching bud morphology and differentiation, physiology and moleculargenetics in sexual special ramie. This research could promote to ulitize the heterosis and improve on thebreeding method.
About the studis on sexual determination of ramie, the gynandrous primordium was formed firstlyduring sex differentiation in male flower. Then the male flower could be shaped by degenerated thegynoecium. But the androecium was not appeared in female flower. When the bud growed to 0.5centimeter before the the gynandrous primordium was formed, the male were easily induced by GA_3among apomixes species. The exact time and maker of external morphology that happened at sexualdetermination date in ramie were not known.The study of relation between the exogenous hormone andsex was mostly focus on reversing sex in ramie.It was not reported about the related genes of sexdetermination in ramie.
In this paper, the ramie materials with unusual sex was studied on observing in botany character,researching bud differentiation, mensurating endogenesis ethylene content, spraying the ethyleneinhibitor, cloning the ACC synthase and studying spatio-temporal expression.The main result of thispaper are as follows:
1. The female ramie (GBN-09) and hermaphrodite ramie(GBN-08) were investigated in second crop.
There are light red leafstalk, connate stipul, kelly stem, green bud and small stem spot in GBN-08.it was different with GBN-09 that have green leafstalk, asunder stipul, white yellow bud and bigstem spot.
2. The growth trend was obtained by observing the GBN-09 and GBN-O8.The result showed the daygrowth trends of stem highness was parabola on GBN-08 and GBN-09 in the second crops, wasgradually reduced in third crops. The day growth trends of node numebers was faster ininitiat-stage than late-stage. It is the same growth trends between the stem highness and leaf lengthand leaf width, and the growth trends is positive correlation between the leaf length and leaf width.
3. The characteristic of flowering were studied in female ramie (GBN-09) and hermaphrodite ramie(GBN-08). The result showed that the sex of individual plant of ramie was determined by the fastbud (which was earliest differentiation). If the fast bud was female, that the individual plant oframie was female. But if the the first bud was male, that the individual plant of ramie washermaphrodite.
4. Morphological observation of flower bud differentiation were done with GBN-08 and GBN-09 byparaffin section. It was the time of sex determination in hermaphrodite ramie including 28-30 node When the bud was 0.2±0.067 centimeter; And It was the time of sex determination in female ramieincluding 24-29 node When the bud was 0.25±0.037 centimeter.
5. The ethylene release rate was measured in shoot tip of different sex ramie in different time, the budof same node in female and hermaphrodite ramie in second crops and different sex singleinflorescence in hermaphrodite ramie. The result indicated there was closed relationship betweensexual different ramie and ethylene release rate. The female ramie could be induced by highethylene release rate. the ethylene content in ramie plant with female flower is higher than ramieplant with androgynary in stem apex, leaf and flower. In flower organ, the ethylene content ishigher in female flower than male flower and mixed inflorescence. The sex expression of ramiewas found to be controlled by foliar spray with 2-aminoethoxyvinylglycine(AVG) and AgNO3.AVG at a concentration of 300,100mg/L and AgNO3 at a 100, 300,500mg/L caused increase ofmaleness and inhibition of fe-male flower. AVG at a concentration of 300mg/L were the best.
6. It is necessary to extract total RNA from plant tissue.A new reagent—RNAplant was screened outto extract RNA from ramie leaf, stern, female flower and fruit by used five different reagents.RNAswere effective extracted and the high quality RNA can be used for cDNA synthesis, RT-PCR andothers downstream experiment.
7. ACC synthase(ACS) was a key enzyme for ethylene biosynthesis. In this paper, four degenerateoligonucleotide primers were designed, coding for consernative amino acid regions in ACCsynthase protein family of dicotyledon. Reverse transcription PCR and Nest PCR were performedon total RNA extracted from shoot tip of ramie, and produced 615bp fragment. By using theprogram of BLASTn and BLASTx on NCBI GenBank database, the fragment was ACC synthasegene, coded 205 amino acids. Domain of deduced ACS amino acids in ramie was aligned withACS amino acids of twenty plant. The result showed that there are five conserve domain in ACSamino acids of ramie.The ramie have higher similar with soybean by phylogeny.
8. To study the differential expression of ACS gene at different development stage and in differenttissues of ramie,relatively quantitative analysis was performed by Realtime-PCR. The expressionlevel of ACS gene in stem apex was higher than leaf, stem and lowest in root in seedling. Theexpression level of ACS gene was lower in anthesis and fruiting period. The expression level ofACS gene in stem apex or bud of GBN-09 was higher than that of GBN-08 when size of the firstbud was 0.75 centimeter.
So, the new progress about the sex determination of ramie was obtained in botany character,researching bud differentiation, mensurating endogenesis ethylene content, spraying the ethyleneinhibitor, cloning the ACC synthase and studying spatio-temporal expression.. The results provided aexprerimental and theoretical basis for controling the sex of ramie, utilizing the hybrid vigor andimproving the breeding methods.
引文
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