不同大豆品种叶片含氮代谢物的变化及SSR标记分析
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摘要
大豆是世界五大作物之一,是非常重要的植物蛋白质和食用油资源,也是重要的优质工业原料,世界各国均高度重视大豆的综合利用和开发。大豆的经济价值更大程度上取决于其蛋白质的含量,蛋白质含量的高低是衡量大豆品质优劣的重要指标。我国是栽培大豆的起源地,拥有最为丰富的栽培大豆、野生大豆资源。但作为同属的野生大豆和栽培大豆的蛋白质含量具有较大差异,与两者的蛋白质形成机制有重要的关系。野生大豆具有蛋白质含量高、适应性广、抗逆性强等优点,野生大豆这种高蛋白质的特性正是未来高蛋白大豆育种最需要的性状。
本研究选取三类不同蛋白质含量的大豆品种：高蛋白野生大豆、普通野生大豆和栽培大豆,详细分析了大豆品种在不同生育时期叶片含氮代谢物(硝态氮含量、亚硝态氮含量、游离氨基酸含量和可溶性蛋白质含量)以及氮循环中关键酶—硝酸还原酶活性的变化,并对不同蛋白质含量的大豆品种进行SSR分子标记检测。从生理生化角度和分子生物学角度分析不同品种大豆氮代谢的变化规律及高蛋白野生大豆籽粒高蛋白质含量形成的机制。结果如下：
1、在整个生育过程中,幼苗期硝酸还原酶活性和硝态氮含量最高,到成熟期都达到最低值,二者的变化都是基本呈降低趋势的；亚硝态氮含量在成熟期最高,初荚期最低；游离氨基酸含量以盛花期最高；可溶性蛋白质含量在初荚期时达到最高值。
2、在大豆各个生育时期内,苗期叶片硝酸还原酶活性与籽粒蛋白质含量呈负相关,其它时期均呈现正相关；分枝期的亚硝态氮含量和游离氨基酸含量,盛花期和成熟期的可溶性蛋白质含量都与籽粒蛋白含量成极显著正相关；而硝态氮含量与籽粒蛋白质的含量相关性不明显。
3、筛选出的12对SSR引物从24份大豆材料中共检测到87个等位变异,每个SSR位点的等位变异范围为4～10个,平均为7.2500个；有效等位基因总数为60.2196个,平均值为5.0183个,占观察到等位基因数的69.2%;Shannon-weaver指数变化范围为1.2389～2.1662,平均值为1.6933；Nei期望杂合性最高为0.8750,最低为0.6372,平均值为0.7755。
4、24份大豆材料的遗传相似性系数范围：0.000-0.895,其中ZYD00857和ZYD01251遗传相似性系数最大,为0.895,ZYD05610和ZYD01018最低,为0.000。在遗传相似系数为0.165处,NTSYS软件将24份大豆材料聚类为三大类：第一大类,3份栽培大豆、蛋白含量低于和高于45.4%的野生大豆各2份；第二大类,15份蛋白含量超过了45.4%野生大豆及蛋白含量为45.07%的野生大豆ZYD01040；第三类,一份蛋白含量很低的野生大豆ZYD05610。这说明能够通过以SSR标记为基础建立的聚类树状图将多数不同蛋白质含量的大豆种质区分开来。
Soybean is one of the five worldwide crops. It is a very important resource of plant protein and oil, and also an important quality industrial raw material. The comprehensive utilization and development of soybean are highly valued by countries around the world. The economic value of soybean depends on its protein content which is the important index to measure soybean quality in a greater extent. China, which has the most abundant cultivated and wild soybean, is the origin of cultivated soybean. The wild and cultivated soybean have different protein contents although they belong to the same genus, which have an important relationship to the formation mechanism of protein. Wild soybean has many advantages such as high protein content, wide adaptability, strong resistance, and so on. The high-protein characteristic of wild soybean will be needed most in high-protein soybean breeding in the future.
Three different protein contents of soybeans were selected in this study:high-protein wild soybean, common wild soybean and cultivated soybean. The nitrogen metabolites (nitrate nitrogen content, nitrite nitrogen content, free amino acids content and soluble protein content) at different growth stages in soybean leaves and the variation of the key enzyme-nitrate reductase activity in the nitrogen cycle had been analysed in detail. The soybeans with different protein contents were detected by SSR molecular marker. The variation of nitrogen metabolism in different varieties of soybean and the formation mechanism of high-protein wild soybean kernel's high-protein content were analyzed from the physiological, biochemical and molecular biological perspectives. Results are as follows:
1. In the whole growth process, the nitrate reductase activity and nitrate nitrogen content of the seedling stage were the highest and reached the minimum value at the mature stage. The basic trend of their variation was reduced. The highest nitrite content was at the mature stage while the lowest was at the early pod setting stage; the highest free aminoacid content was at the flowering stage. The soluble protein content reached the highest value at the early pod setting stage.
2. Between nitrate reductase activity of the seedling stage and grain protein content were negative correlation at all growth periods of soybean and they showed positive correlation at other stages. Both the nitrite nitrogen content and free amino acids content at the ramification stage and the soluble protein content at flowering stage and mature stage were extreme significant positive correlation with grain protein content. But nitrate content had no obvious correlation with grain protein content.
3. A total of 87 alleles were detected from 24 soybean materials with 12 pairs of SSR primer that were screened. Each SSR locus's alleles ranged from 4 to 10 with average for 7.2500. The total of effective alleles were 60.2196 with average for 5.0183, which was 69.2% of the observed alleles; Shannon-weaver indexes were in the range of 1.2389～2.1662 with average for 1.6933. The highest value of Nei expected heterozygosity was 0.8750 and the lowest value was 0.6372 with average for 0.7755.
4. The genetic similarity coefficient's range of 24 soybean materials was 0.000-0.895. The largest coefficient of genetic similarity was 0.895 between ZYD00857 and ZYD01251 among 24 soybean materials. The lowest coefficient of genetic similarity was 0.000 between ZYD05610 and ZYD01018.24 soybean materials were clustered into three major categories by NTSYS software at the cutoff of the genetic similarity coefficient 0.165:the first category included 3 cultivated soybeans,2 wild soybeans with protein content under 45.4% and 2 wild soybeans with protein content over 45.4%; the second category included 15 wild soybeans with protein content over 45.4% and the wild soybean ZYD01040 with protein content 45.07%; The wild soybean ZYD05610 with very low protein content was the third category. This shows that the establishment of clustering dendrogram based to SSR marker can be used to distinguish the majority of the soybeans with different protein content.
本研究选取三类不同蛋白质含量的大豆品种：高蛋白野生大豆、普通野生大豆和栽培大豆,详细分析了大豆品种在不同生育时期叶片含氮代谢物(硝态氮含量、亚硝态氮含量、游离氨基酸含量和可溶性蛋白质含量)以及氮循环中关键酶—硝酸还原酶活性的变化,并对不同蛋白质含量的大豆品种进行SSR分子标记检测。从生理生化角度和分子生物学角度分析不同品种大豆氮代谢的变化规律及高蛋白野生大豆籽粒高蛋白质含量形成的机制。结果如下：
1、在整个生育过程中,幼苗期硝酸还原酶活性和硝态氮含量最高,到成熟期都达到最低值,二者的变化都是基本呈降低趋势的；亚硝态氮含量在成熟期最高,初荚期最低；游离氨基酸含量以盛花期最高；可溶性蛋白质含量在初荚期时达到最高值。
2、在大豆各个生育时期内,苗期叶片硝酸还原酶活性与籽粒蛋白质含量呈负相关,其它时期均呈现正相关；分枝期的亚硝态氮含量和游离氨基酸含量,盛花期和成熟期的可溶性蛋白质含量都与籽粒蛋白含量成极显著正相关；而硝态氮含量与籽粒蛋白质的含量相关性不明显。
3、筛选出的12对SSR引物从24份大豆材料中共检测到87个等位变异,每个SSR位点的等位变异范围为4～10个,平均为7.2500个；有效等位基因总数为60.2196个,平均值为5.0183个,占观察到等位基因数的69.2%;Shannon-weaver指数变化范围为1.2389～2.1662,平均值为1.6933；Nei期望杂合性最高为0.8750,最低为0.6372,平均值为0.7755。
4、24份大豆材料的遗传相似性系数范围：0.000-0.895,其中ZYD00857和ZYD01251遗传相似性系数最大,为0.895,ZYD05610和ZYD01018最低,为0.000。在遗传相似系数为0.165处,NTSYS软件将24份大豆材料聚类为三大类：第一大类,3份栽培大豆、蛋白含量低于和高于45.4%的野生大豆各2份；第二大类,15份蛋白含量超过了45.4%野生大豆及蛋白含量为45.07%的野生大豆ZYD01040；第三类,一份蛋白含量很低的野生大豆ZYD05610。这说明能够通过以SSR标记为基础建立的聚类树状图将多数不同蛋白质含量的大豆种质区分开来。
Soybean is one of the five worldwide crops. It is a very important resource of plant protein and oil, and also an important quality industrial raw material. The comprehensive utilization and development of soybean are highly valued by countries around the world. The economic value of soybean depends on its protein content which is the important index to measure soybean quality in a greater extent. China, which has the most abundant cultivated and wild soybean, is the origin of cultivated soybean. The wild and cultivated soybean have different protein contents although they belong to the same genus, which have an important relationship to the formation mechanism of protein. Wild soybean has many advantages such as high protein content, wide adaptability, strong resistance, and so on. The high-protein characteristic of wild soybean will be needed most in high-protein soybean breeding in the future.
Three different protein contents of soybeans were selected in this study:high-protein wild soybean, common wild soybean and cultivated soybean. The nitrogen metabolites (nitrate nitrogen content, nitrite nitrogen content, free amino acids content and soluble protein content) at different growth stages in soybean leaves and the variation of the key enzyme-nitrate reductase activity in the nitrogen cycle had been analysed in detail. The soybeans with different protein contents were detected by SSR molecular marker. The variation of nitrogen metabolism in different varieties of soybean and the formation mechanism of high-protein wild soybean kernel's high-protein content were analyzed from the physiological, biochemical and molecular biological perspectives. Results are as follows:
1. In the whole growth process, the nitrate reductase activity and nitrate nitrogen content of the seedling stage were the highest and reached the minimum value at the mature stage. The basic trend of their variation was reduced. The highest nitrite content was at the mature stage while the lowest was at the early pod setting stage; the highest free aminoacid content was at the flowering stage. The soluble protein content reached the highest value at the early pod setting stage.
2. Between nitrate reductase activity of the seedling stage and grain protein content were negative correlation at all growth periods of soybean and they showed positive correlation at other stages. Both the nitrite nitrogen content and free amino acids content at the ramification stage and the soluble protein content at flowering stage and mature stage were extreme significant positive correlation with grain protein content. But nitrate content had no obvious correlation with grain protein content.
3. A total of 87 alleles were detected from 24 soybean materials with 12 pairs of SSR primer that were screened. Each SSR locus's alleles ranged from 4 to 10 with average for 7.2500. The total of effective alleles were 60.2196 with average for 5.0183, which was 69.2% of the observed alleles; Shannon-weaver indexes were in the range of 1.2389～2.1662 with average for 1.6933. The highest value of Nei expected heterozygosity was 0.8750 and the lowest value was 0.6372 with average for 0.7755.
4. The genetic similarity coefficient's range of 24 soybean materials was 0.000-0.895. The largest coefficient of genetic similarity was 0.895 between ZYD00857 and ZYD01251 among 24 soybean materials. The lowest coefficient of genetic similarity was 0.000 between ZYD05610 and ZYD01018.24 soybean materials were clustered into three major categories by NTSYS software at the cutoff of the genetic similarity coefficient 0.165:the first category included 3 cultivated soybeans,2 wild soybeans with protein content under 45.4% and 2 wild soybeans with protein content over 45.4%; the second category included 15 wild soybeans with protein content over 45.4% and the wild soybean ZYD01040 with protein content 45.07%; The wild soybean ZYD05610 with very low protein content was the third category. This shows that the establishment of clustering dendrogram based to SSR marker can be used to distinguish the majority of the soybeans with different protein content.
引文
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