摘要
本试验以抗晚霜仁用杏(Prunus armeniaca×sibirica)优良单株花器官为试材,比较研究了36株抗晚霜仁用杏单株的抗寒性,并以筛选出的最抗寒优株的雌蕊为试材,探索一套适用于仁用杏雌蕊全细胞蛋白质组分离的双向电泳技术,为深入研究仁用杏抗寒机制、筛选抗寒品种以及进一步进行抗性育种奠定基础。研究结果如下:
1.在28棵抗寒性‘优一’(Prunus armeniaca×sibirica’youyi’)优株中,‘优株25号’花瓣、雄蕊和雌蕊过冷却点、电导率及SOD的隶属函数平均值均为最大,分别为0.3334、0.3334、0.3027。各优株不同花器官的排名位次不同,而综合排名第一的‘优株25号’,其不同花器官排名位次均居第一。
2.在8棵抗寒性‘龙王帽’(Prunus armeniaca×sibirica’longwangmao’)优株中,‘优株31号’花瓣、雄蕊和雌蕊过冷却点、电导率及SOD的隶属函数平均值值均为最大,分别为0.3334、0.3201、0.3334。各优株不同花器官的排名位次不同,‘优株31号’综合排名第一,其不同花器官排名位次均居第一。
3.‘优株25号’、‘优株31号’的田间受冻情况均比同园对照轻。‘优株25号’花瓣和雌蕊的受冻率较其对照分别低9.4%、7.8%;‘优株31号’花瓣和雌蕊的受冻率较其对照分别低12.5%、4.9%;优株与对照间有显著差异(P<0.05)。优株受冻后的坐果率也均优于对照,且差异显著(P<0.05)。从花瓣与雌蕊的受冻率看,‘优株25号’和‘优株31号’的受冻率差异均不显著;从受冻后的坐果率看,‘优株25号’受冻后的坐果率高于‘优株31号’,差异显著(P<0.05)。
4.优株各个花器官的过冷却点温度均低于同品种对照,差异显著(P<0.05),‘优株25号’更是达到了极显著水平(P<0.01),‘优株25号’花瓣、雄蕊、雌蕊的过冷却点温度均比同品种对照低0.9℃,‘优株31号’花瓣、雄蕊、雌蕊的过冷却点比同品种对照分别低0.5℃、0.7℃、0.7℃。‘优株25号’花瓣、雄蕊、雌蕊的过冷却点温度与‘优株31号’相比分别低了0.7℃、0.4℃、0.5℃,二者花瓣、雄蕊在P<0.01水平上有差异,而雌蕊在P<0.05水平上有差异。
5.不同品种花器官的电解质外渗率、MDA含量随温度下降呈不断升高的趋势,可溶性蛋白及SOD酶活性呈先升高后降低的趋势。
在不同温度下优株与对照花器官的电解质外渗率、MDA含量均表现为优株小于对照。两个优株花器官的电解质外渗率及MDA含量均表现为‘优株25号’<‘优株31号’。优株的半致死温度均低于对照,且‘优株25号’的半致死温度低于‘优株31号’。结果与电解质外渗率所得结果相符。
同品种优株与对照花器官SOD酶活性不同,‘优株25号’花瓣、雄蕊、雌蕊SOD最大值分别出现在-6℃、-3℃、-3℃,对照‘优一’花瓣、雄蕊、雌蕊SOD最大值分别出现在-5℃、-3℃、-3℃。且除-6℃雌蕊的SOD值外,不同低温胁迫下‘优株25号’花器官的SOD值均高于对照。‘优株31号’花瓣、雄蕊、雌蕊SOD最大值分别出现在-5℃、-3℃、-3℃,对照‘龙王帽’花瓣、雄蕊、雌蕊SOD最大值分别出现在-4℃、-3℃、-3℃。除-4℃花瓣与-7℃雄蕊和雌蕊的SOD值外,不同低温胁迫下‘优株31号’花器官的SOD值均高于对照。不同优株花器官SOD酶活性不同,表现为‘优株25号’>‘优株31号’。
‘龙王帽’花瓣的可溶性蛋白含量最大值出现在-3℃,其他不同品种花瓣、雄蕊、雌蕊可溶性蛋白含量均在-4℃时达到最大值。低温胁迫下,优株花器官可溶性蛋白的含量均高于对照。‘优株31号’雄蕊、雌蕊在-6℃至-7℃之间的可溶性蛋白含量大于‘优株25号’,其余均表现为‘优株25号’>‘优株31号’。
6.建立了适合仁用杏雌蕊蛋白质组学研究的双向电泳体系,即:用TCA/丙酮法与酚提取法相结合的方法提取雌蕊的蛋白质,用常温震荡裂解2h的方法溶解蛋白干粉,之后采用bradford法对提取的蛋白质进行定量,在此基础上,上样量在100ug,上样体积为370ul。第一向等电聚焦采用pH 5-8,17cm的线性IPG胶条进行蛋白质的分离,等电聚焦结束后,经含有DTT和碘乙酰胺的平衡缓冲液Ⅰ、Ⅱ各平衡15min。第二向SDS-PAGE凝胶电泳采用12%的凝胶浓度对蛋白质进行分离。用高分辨率的硝酸银染色,可获得分辨率和重复性均较好的凝胶图谱。
The test take the floral organs of anti-frost Kernel Apricot(Prunus armeniaca×sibirica) superior strains as test materials, studied cold hardiness of 36 superior strains of kernel apricot to frost hazard,and used the pistil of Kernel Apricot of selected superior strains of the most cold stress as the specimens,explore a set of two dimensional electropHoresis technology that applied to separate the whole-cell proteins of the pistil of Kernel Apricot, for further research mechanism of cold hardiness of Kernel apricot, filtrate cold resistant varieties and further lay the foundation for breeding. The results are as follows:
1. In the 28 (Prunus armeniaca×sibirica’youyi’) 'youyi' superior strains to cold stress, average value of the subordinate function of the supercooling point、the electrolyte leakage、superoxide dismutase activity of the petal, stamen and pistil of'25'is 0.3334、0.3334、0.3027,the lagest one.Rank order of The superior strains about different floral organs is different , and not only comprehensive ranking of ' 25', but also the ranking of different floral organs is first.
2 In the 8 'longwangmao' (Prunus armeniaca×sibirica’longwangmao’) superior strains to cold stress, average value of the subordinate function of the supercooling point、the electrolyte leakage、superoxide dismutase activity of the petal, stamen and pistil of '31' is 0.3334、0.3201、0.3334,the lagest one. Rank order of the superior strains about different floral organs is different , and not only comprehensive ranking of ' 31', but also the ranking of different floral organs is first.
3. The freezing rate of ' 25'and' 31'in the field were lighter than the contrast . The freezing rate of petals and pistils of' 25'is lower than the contrast were 9.4%, 7.8%; the freezing rate of petals and pistils of ' 31' is lower than the contrast were 12.5%, 4.9%; there are significant differences between superior strains and contrast (P <0.05). Fruit set of superior strains after freeze injury was better than contrast, and the difference is significant (P <0.05). From the freezing rate of petals pistil of view, difference of the freezing rate of '25' and ' 31' were not significantly different; from the fruit setting rate of view after freeze injury, the fruit setting rate of '25'was taller than ' 31', the difference was significant (P <0.05).
4. Supercooling point temperature of superior strains of various floral organs is lower than the contrast, the difference was significant (P <0.05), ' 25' reached significant level (P <0.01), supercooling point temperature of petal, stamen, pistil of '25 'is lower than the same varieties, is 0.9℃, the supercooling points temperature of petals, stamen, pistil of ' 31 'is lower than the same varieties ,were 0.5℃, 0.7℃, 0.7℃. supercooling point temperature of petals, stamen, pistil of '25' is lower than ' 31 ',is 0.7℃, 0.4℃, 0.5℃, there are differences at the P <0.01 level to the two of petals, stamens,and pistil is different at the P <0.05 level .
5. With the temperature declining, electrolyte leakage, MDA content of different varieties of flower organs is continuance rising , soluble protein and SOD activity was first increased and then decreased.
The electrolyte leakage、MDA content of superior strains of flower organs is lower than contrast under the different temperature。electrolyte leakage, MDA content of two superior strains of floral organs showed ' 25' <'" 31'. lethal temperatures of superior strains were lower than the contrastl. Results is accordance with the results of electrolyte leakage.
SOD activity is different between the sperior strains and the contrast,maximum SOD valueof petal, stamen , pistil of '25' appeared at -6℃, -3℃, -3℃,maximum SOD activity of petal, stamen , pistil of contrast appeared at -5℃, -3℃, -3℃.In addition to the SOD value of the pistil with -6℃, under the different low temperature stress , the SOD value of floral organs of '25' were higher than contrast.maximum SOD valueof petal, stamen , pistil of '31' appeared at -5℃, -3℃, -3℃,maximum SOD activity of petal, stamen , pistil of contrast appeared at -4℃, -3℃, -3℃.In addition to the SOD value of the petalwith -4℃,and the stamen and pistil with-7℃, under the different low temperature stress ,the SOD value of floral organs of '31' were higher than contrast.SOD activity of diffrent superior strains floral organs is diffrent, showing '25'> '31'.
Soluble protein content of petals of 'longwangmao' is maximum in -3℃, soluble protein content of the others reached maximum at -4℃. under the low temperature stress, soluble protein content of floral organ of superior strains were higher than contrast. soluble protein content of stamen, pistil of ' 31'from -6℃to -7℃is digger than '25', the rest showed ' 25'> '31'.
3. Established a set of two dimensional electropHoresis technology that fit proteomics of the pistil of Kernel Apricot。Namely,using combined the TCA / acetone and pHenol extraction method to distill the pistil proteins,dissolve protein powder with lysis 2h at normal temperature shock method, followed by bradford method determine protein concentration, on this basis, the content of sample 100ug, the sample volume of 370ul. Subsequently, first,isoelectric focusing using pH 5-8,17 cm linear IPG strips for protein separation, after isoelectric focusing using balance bufferⅠ,Ⅱthat each contain DTT and iodoacetamide balance strips 15min. The second ,the SDS-PAGE, using 12% gel concentration , separate protein. The gel stained by silver contained, in the end,the gel map with preferably distinguishability and reproducibility was obtained.
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