摘要
金荞麦(Fagopyrum dibotrys (D.Don) Hara)为蓼科(Polygonaceae)荞麦属(Fagopyrum)多年生草本植物,具有抑制肿瘤、抗癌等功效。由于需求量过大,野生资源过度采挖,以及人类的活动对生态环境的破坏,使野生金荞麦资源濒危,并于1999年列入《国家重点保护野生植物名录(第一批)》中。
荞麦属部分种间存在着显著的自交不亲和性,因而进行荞麦种间常规杂交育种困难较大,野生金荞麦以根茎无性繁殖为主。本研究从60Coγ照射的江苏Ⅱ号金荞麦的根茎的后代中筛选出红茎和红叶突变株系,连续多代对突变株系的稳定性以及生理特征进行研究,对不同突变体纯化株系的染色体和同功酶进行分析,并利用cDNA-SRAP分子标记对差异表达基因片段进行研究。
1农艺性状分析
金荞麦在生长发育过程中,红茎和红叶的生育进程受到不同程度的推迟,扦插苗的推迟时间长于根繁苗。绿茎的株高较高,红茎较低且分枝数较多,红叶的叶面积较大。金荞麦根茎在7月-8月中旬进入快速增长期,根茎的干鲜重于10月份达到最高。绿茎的根茎干鲜重较高,红叶略低,与红茎差异显著。绿茎和红茎的分支数以及茎节数与根茎产量形成为负相关关系。连续两年的观察表明农艺性状和生育期趋于稳定。
2抗氧化酶活性和同功酶谱研究
金荞麦叶片的SOD、POD、APX和CAT酶活性随着植物的生长而逐渐升高。红茎和红叶的酶活性在苗期和生长期的高于绿茎株系,但是差异不显著。
金荞麦叶片POD同功酶在不同时期和不同株系间各不一样。红叶远端谱带比红茎和绿茎多出2条；绿茎和红叶的近端酶谱带苗期时为3条,多出红茎1条；生长期绿茎为4条,比红茎和红叶多出1条；花期时都为4条带,绿茎的1条谱带的位置和红茎、红叶不一致。SOD同功酶和CAT同功酶谱带无差异。
连续几代的试验表明金荞麦的酶活性和同功酶谱在不同代之间表现出一定的规律性,金荞麦的遗传趋于稳定。
3光合特性研究
金荞麦叶片中光合色素含量在苗期和花期较低,生长期较高。绿茎中的叶绿素含量较高,红叶中的叶绿素含量在苗期时逐渐超过红茎。Chla/Chlb的比值在苗期较低,生长期较高,花期时有所降低；红叶的Chla/Chlb比值在苗期和生长期都较高。红茎和红叶中的类黄酮和花青素含量都高于绿茎。红茎和绿茎中的类黄酮和花青素含量随着植株的生长逐渐升高,红叶中类黄酮含量随植株的生长逐渐升高,花青素含量随着植株的生长逐渐下降。
金荞麦叶片的净光合速率在生长期较高,苗期和开花期较低。苗期时,绿茎叶片的净光合速率和表观羧化效率较高,红叶叶片中的花青素的存在可能抑制了红叶叶片的光合作用。生长期时,金荞麦的净光合速率日变化均呈双峰曲线,由于气孔限制的原因,具有不同程度的“光合午休”现象。
红茎和红叶在苗期和生长期的Fv/Fm、qP、ΦPSⅡ和ETR等叶绿素荧光动力学参数低于绿茎,而NPQ值却高于绿茎株系。花期时,绿茎的NPQ升高,qP和中PSⅡ也降低,这可能与金荞麦各植株衰老程度有关。各荧光参数的变化,表明辐照后对突变体株系的光合能力有一定的影响,导致红叶和红茎的光合作用较弱。
连续两年的试验,金荞麦中的光合色素含量、光合作用和叶绿素荧光的变化趋势基本上一致,遗传已经相对稳定。
4染色体核型分析
金荞麦染色体的核型均为2A型。绿茎的染色体核型组成为2n=2x=16=12m+4sm,红茎的染色体的核型组成为2n=2x=16=10m+6sm(2S AT),红叶的染色体核型组成为3n=3x=24=15m+9sm(3SAT)。
5有效成分含量研究
金荞麦根茎中的(-)-表儿茶素含量在7-8月份快速增加。红叶金荞麦根茎中的(-)-表儿茶素含量一直高于绿茎,而红茎的含量较低。收获时绿茎根茎中(-)-表儿茶素含量为0.034%,红叶为0.038%,而红茎为0.021%。扦插苗的根茎中有效成分的含量稍低于根繁苗的含量。
6cDNA-SRAP研究
通过改良的CTAB法提取叶片中的总RNA,经反转录后,用RT-PCR直接进行SRAP扩增,分析金荞麦基因的差异表达。从49对引物组合中的筛选出的26对引物对3个样品的cDNA进行扩增,获得576条长度在30-1000bp的条带。其中,特异性条带240条,占总条带数的41.7%,对其中18条特异性差异条带克隆分析,发现突变体中有2条片断功能类似,其中1条可能与天冬氨酸氨基转移酶有关。
Buckwheat (Fagopyrum dibotrys (D. Don.) Hara.) belongs to Fagopyrum genus of Polygonaceae family, its rhizome have function on tumor inhibition and anticancer. It had been one of the "list of national key conservative wild plants in China (the first group)" because of destruction by blind harvest and environment pollution. Buckwheat propagates mainly by asexual mode, and it becomes difficult create new genotypes by traditional breeding.
Buckwheat rhizomes of Jiangsu Ⅱ were irradiated by gamma ray.Red stem and red leaves mutants were induced in M2generation. Agronomic traits, photosynthesis, antioxidative enzymes and (-) epicatechin of selected green and mutants were studied in the paper during2008to2009, and isoenzyme and chromosome were researched as well, and cDNA-SRAP technology was applied for genetic diversity and differential gene expression. Results showed as followed:
(1) The growth process of mutants was retarded, and it was longer in plants propagation by cuttings than propagation from rhizome. The rhizome rapid growth was between July and August, and reached higher dry and wet weight in October. The plant height of green stem buckwheat was the highest, and red stem had the most branches, while the red leaves buckwheat leaf area were the biggest. Root yield of green stem buckwheat was higher than red stem and the difference reached extreme significant level. Branches and main stem of green and red stem have negative correlation with root yield. The results showed agronomic traits and growth process were almost stability heredity.
(2) The activity of SOD, POD, CAT and APX increased accompany with plant growth, the activity of mutants were higher than green stem during seeding stage and growth stage. Two additional bands was found in distal isozymogram of POD of red leaves, while there were three bands on proximal during seedling stage in red leaves and green stem, but red stem had two bands; During growth stage, the green stem had four bands; while the mutant has three during flowering stage,; all had four bands, but one of the green stem had different mobility. The bands number of SOD and CAT was unchanged.
The results showed antioxidative enzymes and isozymogram changed regularly among different descends, which indicated that heredity of the mutants were stability.(3)The photosynthetic pigments were higher during growth stage, and lower during seedling stage and flowering stage. The pigments of green leaves were higher than mutants. The ratio of Chla/Chlb varied as'low-high-low', and the red leaves were the highest during seedling stage and growth stage. Anthocyanin content and flavonoids in the green and red stem were always increasing, while anthocyanin content of red leaves were decreased, and flavonoids varied as red stem, but the anthocyanin content and flavonoids in the green were lower.
Pn of green stems was higher than mutants, and it reached significantly during seedling stage and decreased dramatically during flower period. During seedling stage, Pn and CE of green stem were higher than mutants and increased as plant growth. Anthocyanin content of red leaves had negative correlation with Pn, CE and Fv/Fm, which indicated the existed of anthocyanin suppressed the red leaves photosynthesis. The diurnal net photosynthetic rate exhibited double-peak pattern during growth stage, which was caused principally by stomatal limitation and midday depression were appeared. The research indicated mutants had lower photosynthesis capacity than original plants.
The Fv/Fm, qP,ΦPSⅡ and ETR of mutants were lower, and the red stems were lowest, and NPQ of red stems were highest while the green stems were lowest during seedling stage and growth stage. But during flowering stage, NPQ of green stems increased and qP and ΦPSⅡ lowered, this may be induced by leaves senescence.
The trend of photosynthetic pigments and photosynthetic characteristics and chlorophyll fluorescence varied as the same from2008to2009, which indicated the heredity were stability.
(4) The results of karyotype analysis showed that the green stem was2n=2x=16=12m+4sm, red stem was2n=2x=16=10m+6sm(2SAT), and the red leaves was3n=3x=24=15m+9sm(3SAT). All was belong to2A type, according to the Stebbins' karyotypic classification method.
(5) Active ingredient of buckwheat accumulated swiftly in June and August. Active ingredients in red leaves were higher than green and red stems, and red stems were lowest. It reached0.034%in green stems and0.038%in red leaves and0.021%in red stems. Active ingredient in plants propagation from cuttings was lower than propagation from rhizome.
(6) The RT-PCR and Sequence-related amplified polymorphism (SRAP) was used for analysing differential gene expression.26primer pairs were selected from49primer pairs, and amplified576polymorphic bands with an average of41.7%polymorphic bands per primer pair. Part differential expression bands were cloned and analysised, two special cDNA fragments were found in mutant, and one band had relationship with aspartate aminotransferase.
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