豆科植物蒺藜苜蓿发育相关基因
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摘要
豆科(Leguminosae)是种子植物第三大科,用途广泛,栽培历史长,是人类和动物食物和营养来源的最重要的科属之一。豆科植物与根瘤菌之间的共生固氮系统是生物圈中氮循环的一个主要氮源。与其他豆科物种相比,蒺藜苜蓿(Medicago truncatula)具有遗传转化效率高、生长周期短、染色体数目少、基因组小、严格自花授粉、固氮等特点,已成为豆科生物学和基因组学研究的新型模式植物。十多年来,已建立了相关的蒺藜苜蓿基因组生物信息学公共数据库,发展了一系列能基因组研究工具和遗传突变体库。越来越多的研究已经证明,小RNA在植物生长发育过程中扮演着重要的角色。目前,小RNA(small RNAs)研究最广泛的是microRNA(miRNA)和small interfering RNA (siRNA)。miRNA主要为生物体内源型小RNA; siRNA分为内源与外源型小RNA,其中内源型siRNA主要调控生物体的机能,而外源型siRNA主要是RNAi实验进行基因抑制的手段。内源型siRNA的形成机制已比较清楚,其中TAS3 ta-siRNA是2005年才发现的一种新型调控途径,在生长发育等各种生物学过程中发挥重要作用。本文从蒺藜苜蓿Tntl突变体库中筛选到了两种发育相关突变体：(1)叶缘深锯齿突变体loll.从中克隆到与植物形态发育密切相关基因LOL1,它是植物内源siRNA的TAS3 ta-siRNA调控途径中的关键基因,对生长发育有直接或间接的影响；
(2)滞绿突变体sgr。从中克隆到叶绿素降解相关基因SGR,并且利用RNAi技术抑制牧草紫花苜蓿中内源SGR基因的表达,发现转基因株系滞绿并且品质有所改良；主要研究内容和结果如下：
1. LOL1基因与TAS3 ta-siRNA途径在蒺藜苜蓿发育过程中的作用
在筛选蒺藜苜蓿Tntl突变体库时,发现了四个具相同表型的发育突变体lobed leafletl(loll),在叶、花和根的发育过程中表现出严重缺陷：成年叶边缘产生严重的缺刻,幼年叶呈现出成年叶的特征；花器官的发育也不正常,其花瓣呈放射状,中央心皮不能融合从而导致胚珠暴露,花粉囊和花粉颗粒大小不均一,花粉内染色质发生降解,在开花后期,花粉囊无法正常打开；主根长度变短,但一级和二级侧根的数量明显增多。进一步分析显示,这四个突变体分别是Tntl和内源逆转录转座子Merel插入到同一个基因外显子的不同位置造成的；我们克隆了该基因,并命名为LOU1。
LOL1在蒺藜苜蓿野生型的各器官中均有表达,其中花和茎中表达量最高,其次是叶柄、幼叶和成年叶,这表明该基因在叶和花的发育过程中起到重要作用。RNA原位杂交结果证明,营养生长阶段,LOL1基因在叶原基和处于发育阶段的叶近轴端部位表达,而在茎顶端分生组织(shoot apical meristems, SAM)中没有表达；生殖生长阶段,LOL1在花分生组织有表达,其中在发育中的花药、胚珠和心皮等花器官中表达量更高。
lol1突变体的互补系的发育缺陷被完全恢复,并且能够产生后代。qRT-PCR分析表明外源引入的LOL1基因在互补系中的表达量与野生型相似。过表达LOL1的蒺藜苜蓿转基因株系的表型和野生型没有明显差别。
系统进化树分析表明LOL1氨基酸序列与拟南芥AGO7(ZIP)、水稻OsAGO7(SHL4)有高的同源性,聚为一组。另外,LOL1含有AGO蛋白的PAZ中心结构域和C-端PIWI结构域,表明是拟南芥AGO7的同源基因,其编码的蛋白是TAS3ta-siRNA途径的关键蛋白。
通过对蒺藜苜蓿TAS3 ta-siRNA途径的预测和分析,得到了蒺藜苜蓿TAS3ta-siRNA基因,TAS3 ta-siRNA途径中的miRNA-miR390,和三个靶基因ARF。结果表明,这一途径的关键基因序列都与拟南芥、百脉根和水稻中的相关基因同源,且靶位点保守。这些结果表明TAS3 ta-siRNA-ARFs在调控单子叶和双子叶植物发育过程中的功能非常保守。而且,蒺藜苜蓿loll突变体特有的根系统缺陷表明TAS3 ta-siRNA调控途径在蒺藜苜蓿发育中可能发挥更加广泛的作用。另外,HD-ZIPⅢ基因家族在lol1突变体中表达上调,这说明该基因家族被蒺藜苜蓿TAS3 ta-siRNA途径间接调控。由于该基因家族控制植物侧翼器官的发育,因此HD-ZIPⅢ基因家族表达水平的变化可能也是造成loll突变体侧根数目增多的原因之一。
2.蒺藜苜蓿滞绿基因(STAY-GREEN)拘鉴定
本实验从蒺藜苜蓿Tntl反转录转座子突变体库中还筛选到了滞绿(stay-green)突变体sgr,在叶片衰老的过程中仍保留叶绿素。研究发现Tntl插入到了蒺藜苜蓿的滞绿(STAY-GREEN)基因MtSGR的第三个外显子第131-132bp之间,导致基因失活。根据蒺藜苜蓿sgr突变体在衰老过程中叶绿素的保留、Fv／Fm指标下降速率和类囊体结构保留的超微结构观察结果,证明该突变体属于C型滞绿突变体。
系统进化树结果表明蒺藜苜蓿SGR(MtSGR)与紫花苜蓿(MsSGR)亲缘关系最近。而且豌豆、大豆、紫花苜蓿、蒺藜苜蓿等豆科物种的SGR序列高度保守,聚为一个组。蛋白质序列比对显示,SGR家族成员蛋白质有高度保守的中心序列,但N端和C端序列区域有显著差别。蒺藜苜蓿sgr突变体中Tntl插入位点Arg-145在SGR家族内部是一个高度保守的氨基酸残基,说明该氨基酸是蛋白质具有功能的保守氨基酸。
MtSGR基因表达谱结果显示,其在各器官中均为低水平组成型表达,但在种子成熟与根瘤发育,特别是在根瘤衰老的过程中,MtSGR的转录水平明显升高,表明MtSGR的表达不仅与叶绿素衰老相关,在根瘤发育和衰老途径中也可能发挥功能。
3.紫花苜蓿内源SGR基因RNAi分析
根据蒺藜苜蓿与紫花苜蓿间高度同源的进化关系,克隆了紫花苜蓿MsSGR基因,并且利用RNAi技术对紫花苜蓿内源MsSGR基因的表达进行抑制,获得了12个滞绿转基因株系。通过real-time PCR分析,发现5个转基因株系内源SGR基因的抑制水平在60%以上。衰老诱导实验表明,这5个转基因株系表型与蒺藜苜蓿sgr突变体一致,属于C型滞绿突变体。Southern杂交结果表明,这5个转基因株系为独立的转基因事件,转基因稳定地整合到了紫花苜蓿基因组的不同区域。
紫花苜蓿是一种重要的牧草,收割方式通常是将地上部分采收后捆包,晾干成干草。干草是目前最普遍的贮存草料的方法,能够避免在收后贮藏时候草料质量的损失。对紫花苜蓿转基因株系进行模拟收割和干燥,发现在正常生长环境下转基因株系与野生型、对照株系没有区别,但在干燥处理之后仍保持滞绿的特点,因此在收割与收割后贮存上有明显优势和潜在的应用价值。
牧草品质分析表明,与野生型相比,转基因株系的牧草品质没有降低,甚至有一定的改进,比如SGRi-39株系的粗蛋白含量和消化度稍有少许提高,而耐酸性去污剂纤维(ADF)和中性去污剂纤维(NDF)含量则明显下降,说明MsSGR的下调表达对紫花苜蓿的牧草改良有正面贡献。
综上所述,我们首次将滞绿(stay-green)性状应用到了豆科牧草中,证明叶绿素降解途径中的滞绿基因SGR可成功用于牧草紫花苜蓿的遗传改良。
Leguminosae, the third largest family of flowing plants, grows throughout the world, and is of great economic importance for timber, fodder, drugs, and food. As for many legume species, unique advantage is their response to nitrogen limitation-nitrogen fixation from the soil by the formation of nodules, resulting in the symbiosis with rhizobia. It can reduce the fertilizer costs in agriculture. However, the genome evolution of many legume species is closely tied with history of human civilization, which made the genome duplicated and even redundant.
Currently Medicago truncatula is being used as a model of legume plants since it has a modest diploid genome, self-fertile nature, relatively short generation cycle, close relationships to alfalfa and other forage legumes, and large collections of ecotypes. The genome sequencing work of Medicago truncatula has been largely developed in the past decade. A growing number of genome websites and database as well as excellent mutant populations have already been set up, which accelerates the work of M. truncatula functional genomics. As a result, the supplications of genome and transcriptome information of M. truncatula have stimulated to understand the genetics and development mechanisms in alfalfa and other legume crops.
Small RNAs have been proved to play important roles in plant development. Until now, micro RNA (miRNA) and small interfering RNA (siRNA) are two kinds of small RNAs which have been well studied. microRNA is a native RNA regulator in plants. The formation of siRNAs can be from endogenous pathway and transfection of exogenous double-strand RNA. A new type of endogenous TAS3 ta-siRNA pathway was reported in Arabidopsis and rice in 2005. Exogenous siRNAs are usually used as a tool to study the target gene function, which is commonly utilized in RNAi technique.
Here we have identified and characterized two kinds of developmental defect mutants-lobed leaflet1 and stay-green-from the tobacco(Nicotiana tabacum) Tnt1 retrotransposon-tagged mutant population of M. truncatula.This thesis describes the identification and molecular characterization of the genes LOL1 and SGR related with the mutant phenotypes.
1. Identification of LOL1 gene and its role in M. truncatula TAS3 ta-siRNA pathway during plant development
In this study, we found four developmental defective mutant lines showing the same phenotypic changes. They were named loll-1 to loll-4(lobed leaflet1) respectively, because all adult leaves are lobed in these mutants. Their juvenile leaves exhibit the characteristics of adult leaves which have trifoliate form. Moreover, further analysis showed abnormal changes in flower and roots. The keel and alae split with each other and unfused central carpels resulted in the exposed ovules. The size of pollen and anther sacs was not even and some pollens were dead. During flower stage, the anthers could not open, failing in the fertilization. Observation of 12-day-old roots showed the increasing number of first and secondary order lateral roots but shorter primary roots in loll mutants compared with wild-type.
LOL1 complementation and over-expression transgenic lines showed the same phenotype as that of wild-type. This result, combining with the analysis of back-crossing revealed the abnormal phenotypes was tightly linked to the LOL1 which is a single recessive locus.
Phylogenetic analysis showed that LOL1 was clustered into a subgroup including the AGO7 (ZIP) in Arabidopsis and OsAGO7 (SHL4) in rice with the high similarity. In addition, LOL1 contained a central PAZ domain and a C-terminal PIWI domain which define the PPD class of proteins, also named as AGO proteins. Therefore, LOL1 is an ortholog of Arabidopsis ARGONAUTE7, which is a key gene involved in TAS3 ta-siRNA pathway.
By prediction and analysis of M. truncatula TAS3 ta-siRNA pathway, we analyzed Mt miR390 cleavage manner, the TAS3 ta-siRNA genes and target genes ARFs. The result revealed that TAS3 ta-siRNA biogenesis mechanism is conserved in Medicago truncatula. These data indicate that the regulation mechanism of TAS3 tasiRNA-ARFs pathway is well conserved in both monocots and dicots and plays a fundamental role in plant development.
In our study, the severe defects in both primary and lateral roots in lollmutants suggest that the root development is also sensitive to the TAS3 ta-siRNA pathway. Our data indicate that besides three ARF genes directly regulated, HD-ZIP III gene family are also regulated indirectly by TAS3 ta-siRNA pathway in root of M. truncatula. Compared with no root phenotypic changes observed in Arabidopsis ago7 mutant, defective roots of M. truncatula loll mutant showed wider range of regulation in M. truncatula development.
2. Identification of M. truncatula STAY-GREEN(SGR) gene
A stay-green(sgr) mutant line NF2089 was obtained by screening the tobacco Tntl-tagged mutant population of M. truncatula. The whole plants remained green during natural and dark-induced senescence. Interestingly, the stay-green phenomenon was evident not only in leaves but also in other organs, such as anther, central carpels, mature pods and seeds. After 10 days of dark-induced senescence, chlorophyll (Ch1) contents were retained much in NF2089,60% of Ch1 a and 58% of Ch1 b retained respectively. Combining with the normal decrease of the maximal fluorescence yield of photosystem II (PSII)-Fv/Fm value and retaining of thylakoid structure in senescent chloroplasts, M. truncatula sgr mutant belongs to type C stay-green mutant.
SGR was cloned from M. truncatula and M. sativa SGR, the phylogenetic trees based on predicted SGR protein sequences were constructed, showing that MtSGR and MsSGR were most identical to each other, with closely similarity to PsSGR. All the SGRs from Leguminosae species including soybean, pea, M. sativa and M. truncatula were clustered together. The alignment analysis revealed that SGR family members share highly conserved central core with divergent at N and C termini. The amino acid residue Arg-145, at which the Tntl was inserted into NF2089, is an invariant residue within SGR family.
MtSGR gene was constitutively expressed in all organs at low level. It is noteworthy, however, transcription was highly induced during seed maturation and nodule development with a dramatically improvement during nodule senescence. Therefore, it suggested that MtSGR is not only related with chlorophyll senescence, but may be also involved in nodule development and senescence.
Altogether, we identified MtSGR gene, which is member of SGR gene family, from M. truncatula sgr mutant with stable type C stay-green phenotype during senescence. Based on the gene expression atlas, we propose that MtSGR is performed in nodule development and senescence pathway, suggesting overlap between chlorophyll senescence and nodule development.
3. Down-regulation of MsSGR in alfalfa by RNA interference
Alfalfa is regarded as important forage around the world. Hay is the most common method of forage preservation for animal feed and storage, which can cause little loss of forage material. When alfalfa in field is used as hay, the harvesting is usually fresh-cut, baled and then dried. In order to improve the quality of alfalfa, it is also important to breed stay-green cultivar whose leaf senescence can be delayed. Therefore, we cloned SGR from alfalfa (M. sativa) according to the conserved relationship between M. truncatula and alfalfa. To analyze the role of MsSGR, the MsSGR-RNAi transgenic lines of alfalfa was constructed through transformation of MsSGR-RNAi vector under the control of 35S-promoter. Five out of twelve positive transgenic lines, MsSGR-RNAi (SGRi) No.10,17,21,29, and 39 have significantly lower SGR transcription level by more than 60% compared with control line (CTRL) using qRT-PCR. The analysis of dark-induced senescence showed that the stay-green phynotype of RNAi transgenic lines also showed the characterization of type C stay-green mutant.
To evaluate the potential application of MsSGR-RNAi alfalfa, we performed the mimic harvest process of these transgenic alfalfa lines. overground parts of each transgenic line and wild-type were cut and put in the field where the air is well ventilated. During the naturally dry progress, transgenic lines still stably showed the stay-green phenotype, compared with the yellowing of wild-type. Therefore, transgenic alfalfa lines have the potential predominance in harvest and post-harvest storage.
We evaluated all the five MsSGR-RNAi transgenic alfalfa lines by forage analysis. It was found that No.39 has higher contents of crude protein, lower in value of ADF and NDF, compared with wild-type RSY-4D. It is commonly valued that forages with higher digestibility could supply more energy to the animal per unit of DM consumed than less digestible forages could. So the strategy of senescence delay leads to forage improvements as predicted above.
In this part of study, we identified the homologs of SGR in forage alfalfa (M. sativa), and obtained stay-green transgenic alfalfa by RNA interference silencing of SGR expression. Stay-green phenotype was remained even after plants were cut off and dried. By the senescence inhibition, the forage quality analysis of transgenic alfalfa lines shows no loss but a slightly improvement, which might be considered a good sign for application of stay-green phenotype. Our work also constructs a bridge between the theoretical study of genes in M. truncatula and application of genetic improvement of alfalfa. There are few comments about alfalfa improvement of delaying leaf senescence by directly genetic manipulation. And this is the first report on applications of stay-green into legume forages.
(2)滞绿突变体sgr。从中克隆到叶绿素降解相关基因SGR,并且利用RNAi技术抑制牧草紫花苜蓿中内源SGR基因的表达,发现转基因株系滞绿并且品质有所改良；主要研究内容和结果如下：
1. LOL1基因与TAS3 ta-siRNA途径在蒺藜苜蓿发育过程中的作用
在筛选蒺藜苜蓿Tntl突变体库时,发现了四个具相同表型的发育突变体lobed leafletl(loll),在叶、花和根的发育过程中表现出严重缺陷：成年叶边缘产生严重的缺刻,幼年叶呈现出成年叶的特征；花器官的发育也不正常,其花瓣呈放射状,中央心皮不能融合从而导致胚珠暴露,花粉囊和花粉颗粒大小不均一,花粉内染色质发生降解,在开花后期,花粉囊无法正常打开；主根长度变短,但一级和二级侧根的数量明显增多。进一步分析显示,这四个突变体分别是Tntl和内源逆转录转座子Merel插入到同一个基因外显子的不同位置造成的；我们克隆了该基因,并命名为LOU1。
LOL1在蒺藜苜蓿野生型的各器官中均有表达,其中花和茎中表达量最高,其次是叶柄、幼叶和成年叶,这表明该基因在叶和花的发育过程中起到重要作用。RNA原位杂交结果证明,营养生长阶段,LOL1基因在叶原基和处于发育阶段的叶近轴端部位表达,而在茎顶端分生组织(shoot apical meristems, SAM)中没有表达；生殖生长阶段,LOL1在花分生组织有表达,其中在发育中的花药、胚珠和心皮等花器官中表达量更高。
lol1突变体的互补系的发育缺陷被完全恢复,并且能够产生后代。qRT-PCR分析表明外源引入的LOL1基因在互补系中的表达量与野生型相似。过表达LOL1的蒺藜苜蓿转基因株系的表型和野生型没有明显差别。
系统进化树分析表明LOL1氨基酸序列与拟南芥AGO7(ZIP)、水稻OsAGO7(SHL4)有高的同源性,聚为一组。另外,LOL1含有AGO蛋白的PAZ中心结构域和C-端PIWI结构域,表明是拟南芥AGO7的同源基因,其编码的蛋白是TAS3ta-siRNA途径的关键蛋白。
通过对蒺藜苜蓿TAS3 ta-siRNA途径的预测和分析,得到了蒺藜苜蓿TAS3ta-siRNA基因,TAS3 ta-siRNA途径中的miRNA-miR390,和三个靶基因ARF。结果表明,这一途径的关键基因序列都与拟南芥、百脉根和水稻中的相关基因同源,且靶位点保守。这些结果表明TAS3 ta-siRNA-ARFs在调控单子叶和双子叶植物发育过程中的功能非常保守。而且,蒺藜苜蓿loll突变体特有的根系统缺陷表明TAS3 ta-siRNA调控途径在蒺藜苜蓿发育中可能发挥更加广泛的作用。另外,HD-ZIPⅢ基因家族在lol1突变体中表达上调,这说明该基因家族被蒺藜苜蓿TAS3 ta-siRNA途径间接调控。由于该基因家族控制植物侧翼器官的发育,因此HD-ZIPⅢ基因家族表达水平的变化可能也是造成loll突变体侧根数目增多的原因之一。
2.蒺藜苜蓿滞绿基因(STAY-GREEN)拘鉴定
本实验从蒺藜苜蓿Tntl反转录转座子突变体库中还筛选到了滞绿(stay-green)突变体sgr,在叶片衰老的过程中仍保留叶绿素。研究发现Tntl插入到了蒺藜苜蓿的滞绿(STAY-GREEN)基因MtSGR的第三个外显子第131-132bp之间,导致基因失活。根据蒺藜苜蓿sgr突变体在衰老过程中叶绿素的保留、Fv／Fm指标下降速率和类囊体结构保留的超微结构观察结果,证明该突变体属于C型滞绿突变体。
系统进化树结果表明蒺藜苜蓿SGR(MtSGR)与紫花苜蓿(MsSGR)亲缘关系最近。而且豌豆、大豆、紫花苜蓿、蒺藜苜蓿等豆科物种的SGR序列高度保守,聚为一个组。蛋白质序列比对显示,SGR家族成员蛋白质有高度保守的中心序列,但N端和C端序列区域有显著差别。蒺藜苜蓿sgr突变体中Tntl插入位点Arg-145在SGR家族内部是一个高度保守的氨基酸残基,说明该氨基酸是蛋白质具有功能的保守氨基酸。
MtSGR基因表达谱结果显示,其在各器官中均为低水平组成型表达,但在种子成熟与根瘤发育,特别是在根瘤衰老的过程中,MtSGR的转录水平明显升高,表明MtSGR的表达不仅与叶绿素衰老相关,在根瘤发育和衰老途径中也可能发挥功能。
3.紫花苜蓿内源SGR基因RNAi分析
根据蒺藜苜蓿与紫花苜蓿间高度同源的进化关系,克隆了紫花苜蓿MsSGR基因,并且利用RNAi技术对紫花苜蓿内源MsSGR基因的表达进行抑制,获得了12个滞绿转基因株系。通过real-time PCR分析,发现5个转基因株系内源SGR基因的抑制水平在60%以上。衰老诱导实验表明,这5个转基因株系表型与蒺藜苜蓿sgr突变体一致,属于C型滞绿突变体。Southern杂交结果表明,这5个转基因株系为独立的转基因事件,转基因稳定地整合到了紫花苜蓿基因组的不同区域。
紫花苜蓿是一种重要的牧草,收割方式通常是将地上部分采收后捆包,晾干成干草。干草是目前最普遍的贮存草料的方法,能够避免在收后贮藏时候草料质量的损失。对紫花苜蓿转基因株系进行模拟收割和干燥,发现在正常生长环境下转基因株系与野生型、对照株系没有区别,但在干燥处理之后仍保持滞绿的特点,因此在收割与收割后贮存上有明显优势和潜在的应用价值。
牧草品质分析表明,与野生型相比,转基因株系的牧草品质没有降低,甚至有一定的改进,比如SGRi-39株系的粗蛋白含量和消化度稍有少许提高,而耐酸性去污剂纤维(ADF)和中性去污剂纤维(NDF)含量则明显下降,说明MsSGR的下调表达对紫花苜蓿的牧草改良有正面贡献。
综上所述,我们首次将滞绿(stay-green)性状应用到了豆科牧草中,证明叶绿素降解途径中的滞绿基因SGR可成功用于牧草紫花苜蓿的遗传改良。
Leguminosae, the third largest family of flowing plants, grows throughout the world, and is of great economic importance for timber, fodder, drugs, and food. As for many legume species, unique advantage is their response to nitrogen limitation-nitrogen fixation from the soil by the formation of nodules, resulting in the symbiosis with rhizobia. It can reduce the fertilizer costs in agriculture. However, the genome evolution of many legume species is closely tied with history of human civilization, which made the genome duplicated and even redundant.
Currently Medicago truncatula is being used as a model of legume plants since it has a modest diploid genome, self-fertile nature, relatively short generation cycle, close relationships to alfalfa and other forage legumes, and large collections of ecotypes. The genome sequencing work of Medicago truncatula has been largely developed in the past decade. A growing number of genome websites and database as well as excellent mutant populations have already been set up, which accelerates the work of M. truncatula functional genomics. As a result, the supplications of genome and transcriptome information of M. truncatula have stimulated to understand the genetics and development mechanisms in alfalfa and other legume crops.
Small RNAs have been proved to play important roles in plant development. Until now, micro RNA (miRNA) and small interfering RNA (siRNA) are two kinds of small RNAs which have been well studied. microRNA is a native RNA regulator in plants. The formation of siRNAs can be from endogenous pathway and transfection of exogenous double-strand RNA. A new type of endogenous TAS3 ta-siRNA pathway was reported in Arabidopsis and rice in 2005. Exogenous siRNAs are usually used as a tool to study the target gene function, which is commonly utilized in RNAi technique.
Here we have identified and characterized two kinds of developmental defect mutants-lobed leaflet1 and stay-green-from the tobacco(Nicotiana tabacum) Tnt1 retrotransposon-tagged mutant population of M. truncatula.This thesis describes the identification and molecular characterization of the genes LOL1 and SGR related with the mutant phenotypes.
1. Identification of LOL1 gene and its role in M. truncatula TAS3 ta-siRNA pathway during plant development
In this study, we found four developmental defective mutant lines showing the same phenotypic changes. They were named loll-1 to loll-4(lobed leaflet1) respectively, because all adult leaves are lobed in these mutants. Their juvenile leaves exhibit the characteristics of adult leaves which have trifoliate form. Moreover, further analysis showed abnormal changes in flower and roots. The keel and alae split with each other and unfused central carpels resulted in the exposed ovules. The size of pollen and anther sacs was not even and some pollens were dead. During flower stage, the anthers could not open, failing in the fertilization. Observation of 12-day-old roots showed the increasing number of first and secondary order lateral roots but shorter primary roots in loll mutants compared with wild-type.
LOL1 complementation and over-expression transgenic lines showed the same phenotype as that of wild-type. This result, combining with the analysis of back-crossing revealed the abnormal phenotypes was tightly linked to the LOL1 which is a single recessive locus.
Phylogenetic analysis showed that LOL1 was clustered into a subgroup including the AGO7 (ZIP) in Arabidopsis and OsAGO7 (SHL4) in rice with the high similarity. In addition, LOL1 contained a central PAZ domain and a C-terminal PIWI domain which define the PPD class of proteins, also named as AGO proteins. Therefore, LOL1 is an ortholog of Arabidopsis ARGONAUTE7, which is a key gene involved in TAS3 ta-siRNA pathway.
By prediction and analysis of M. truncatula TAS3 ta-siRNA pathway, we analyzed Mt miR390 cleavage manner, the TAS3 ta-siRNA genes and target genes ARFs. The result revealed that TAS3 ta-siRNA biogenesis mechanism is conserved in Medicago truncatula. These data indicate that the regulation mechanism of TAS3 tasiRNA-ARFs pathway is well conserved in both monocots and dicots and plays a fundamental role in plant development.
In our study, the severe defects in both primary and lateral roots in lollmutants suggest that the root development is also sensitive to the TAS3 ta-siRNA pathway. Our data indicate that besides three ARF genes directly regulated, HD-ZIP III gene family are also regulated indirectly by TAS3 ta-siRNA pathway in root of M. truncatula. Compared with no root phenotypic changes observed in Arabidopsis ago7 mutant, defective roots of M. truncatula loll mutant showed wider range of regulation in M. truncatula development.
2. Identification of M. truncatula STAY-GREEN(SGR) gene
A stay-green(sgr) mutant line NF2089 was obtained by screening the tobacco Tntl-tagged mutant population of M. truncatula. The whole plants remained green during natural and dark-induced senescence. Interestingly, the stay-green phenomenon was evident not only in leaves but also in other organs, such as anther, central carpels, mature pods and seeds. After 10 days of dark-induced senescence, chlorophyll (Ch1) contents were retained much in NF2089,60% of Ch1 a and 58% of Ch1 b retained respectively. Combining with the normal decrease of the maximal fluorescence yield of photosystem II (PSII)-Fv/Fm value and retaining of thylakoid structure in senescent chloroplasts, M. truncatula sgr mutant belongs to type C stay-green mutant.
SGR was cloned from M. truncatula and M. sativa SGR, the phylogenetic trees based on predicted SGR protein sequences were constructed, showing that MtSGR and MsSGR were most identical to each other, with closely similarity to PsSGR. All the SGRs from Leguminosae species including soybean, pea, M. sativa and M. truncatula were clustered together. The alignment analysis revealed that SGR family members share highly conserved central core with divergent at N and C termini. The amino acid residue Arg-145, at which the Tntl was inserted into NF2089, is an invariant residue within SGR family.
MtSGR gene was constitutively expressed in all organs at low level. It is noteworthy, however, transcription was highly induced during seed maturation and nodule development with a dramatically improvement during nodule senescence. Therefore, it suggested that MtSGR is not only related with chlorophyll senescence, but may be also involved in nodule development and senescence.
Altogether, we identified MtSGR gene, which is member of SGR gene family, from M. truncatula sgr mutant with stable type C stay-green phenotype during senescence. Based on the gene expression atlas, we propose that MtSGR is performed in nodule development and senescence pathway, suggesting overlap between chlorophyll senescence and nodule development.
3. Down-regulation of MsSGR in alfalfa by RNA interference
Alfalfa is regarded as important forage around the world. Hay is the most common method of forage preservation for animal feed and storage, which can cause little loss of forage material. When alfalfa in field is used as hay, the harvesting is usually fresh-cut, baled and then dried. In order to improve the quality of alfalfa, it is also important to breed stay-green cultivar whose leaf senescence can be delayed. Therefore, we cloned SGR from alfalfa (M. sativa) according to the conserved relationship between M. truncatula and alfalfa. To analyze the role of MsSGR, the MsSGR-RNAi transgenic lines of alfalfa was constructed through transformation of MsSGR-RNAi vector under the control of 35S-promoter. Five out of twelve positive transgenic lines, MsSGR-RNAi (SGRi) No.10,17,21,29, and 39 have significantly lower SGR transcription level by more than 60% compared with control line (CTRL) using qRT-PCR. The analysis of dark-induced senescence showed that the stay-green phynotype of RNAi transgenic lines also showed the characterization of type C stay-green mutant.
To evaluate the potential application of MsSGR-RNAi alfalfa, we performed the mimic harvest process of these transgenic alfalfa lines. overground parts of each transgenic line and wild-type were cut and put in the field where the air is well ventilated. During the naturally dry progress, transgenic lines still stably showed the stay-green phenotype, compared with the yellowing of wild-type. Therefore, transgenic alfalfa lines have the potential predominance in harvest and post-harvest storage.
We evaluated all the five MsSGR-RNAi transgenic alfalfa lines by forage analysis. It was found that No.39 has higher contents of crude protein, lower in value of ADF and NDF, compared with wild-type RSY-4D. It is commonly valued that forages with higher digestibility could supply more energy to the animal per unit of DM consumed than less digestible forages could. So the strategy of senescence delay leads to forage improvements as predicted above.
In this part of study, we identified the homologs of SGR in forage alfalfa (M. sativa), and obtained stay-green transgenic alfalfa by RNA interference silencing of SGR expression. Stay-green phenotype was remained even after plants were cut off and dried. By the senescence inhibition, the forage quality analysis of transgenic alfalfa lines shows no loss but a slightly improvement, which might be considered a good sign for application of stay-green phenotype. Our work also constructs a bridge between the theoretical study of genes in M. truncatula and application of genetic improvement of alfalfa. There are few comments about alfalfa improvement of delaying leaf senescence by directly genetic manipulation. And this is the first report on applications of stay-green into legume forages.
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