杨树不同种质花粉萌发和致敏蛋白的差异

摘要

[Objective] The characteristics of pollen germination and the differences of allergenic proteins among six poplar germplasms (Populus deltoides '2KEN8',P.deltoides 'Nanyang',P.deltoides × P.maximowiczii 'Eridano',P.deltoides × P.cathayana 'Senhai 2',P.simonii and P.nigra var.italica) were studied to provide a theoretical basis and scientific evidence for breeding hypoallergenic poplar varieties and for the control of plant allergy.[Method] Pollen was collected from flower branches of six poplar germplasms collected from Beijing,Hebei and Henan in March 2015 for a period of hydroponic culture,pollen germination was studied by liquid culture method,and pollen allergenic proteins of different poplar germplasms was used by two-dimensional electrophoresis and real-time quantitative PCR.[Result] 1) The pollen viability of P.deltoides '2KEN8',P.deltoides 'Nanyang' and P.nigra var.italica was higher,followed by P.simonii and P.deltoides × P.maximowiczii 'Eridano',however,the viability of P.deltoides × P.cathayana 'Senhai 2' was the worst;the largest pollen germination rate was in P.deltoides '2KEN8',up to 74.42% (±2.36%),while the pollen germination of triploid P.deltoides × P.cathayana 'Senhai 2' was 0;the average length of pollen tube of P.nigra var.italica was 456 μm (±2.05 μm),the shortest was in P.simonii,276.44 μm (±11.08 μm),and the difference of pollen germination rate and pollen tube length among different entities were highly significant (P<0.01),suggesting that the pollen viability was significantly different among different germplasms.2) The 2-DE maps of P.deltoides 'Nanyang',P.deltoides × P.maximowiczii 'Eridano',P.deltoides × P.cathayana 'Senhai 2',P.simonii and P.nigra var.italica mature pollen were analyzed by Imagemaster 2D platinum 6.0,a total of 408 protein spots were matched.Based on the 28 potentially allergenic proteins of P.deltoides '2KEN8' identified in our previous study,6 allergenic proteins were missing expression in the other five entities;both P.nigra var.italica and P.deltoides 'Nanyang' had 21 allergenic proteins,P.deltoides × P.maximowiczii 'Eridano' had 19;while P.deltoides×P.cathayana 'Senhai 2' and P.deltoides '2KEN8' had the least number of allergenic proteins,only 18.Morover,the analysis of difference expression indicated that 12 of the 22 remaining allergenic proteins were less expressed in the five entities than in P.deltoides '2KEN8',the range of relative expression level was-3.99--0.02.The expression level of P.deltoides × P.cathayana 'Senhai 2' and P.simonii was the least,especially they had 10 and 8 allergenic proteins which were significantly or highly significant lower than that of P.deltoides '2KEN8',respectively.The expression of 3 allergenic proteins in the 5 entities was higher than in P.deltoides '2KEN8'.The strongest expression was in P.deltoides 'Nanyang',significantly greater than in P.deltoides '2KEN8',followed by P.nigra var.italica,P.deltoides × P.cathayana 'Senhai 2',P.deltoides × P.maximowiczii 'Eridano' and P.simonii.Variance analysis among groups suggested that the expression of 17 allergenic proteins of P.nigra var.italica and P.deltoides 'Nanyang' were significantly (P<0.05) or highly significantly (P<0.01) greater than that of P.deltoides × P.cathayana 'Senhai 2' and P.simonii.3) qRT-PCR results showed that there were 5 of 9 allergenic proteins encoding genes highly expressed in P.nigra var.italica and P.deltoides 'Nanyang',including spot 12,51,200,216 and 161,followed by P.simonii,while both of P.deltoides × P.maximowiczii 'Eridano' and P.deltoides × P.cathayana 'Senhai 2' had only 2 highly expressed allergenic protein coding genes.[Conclusion] The pollen viability,the number and expression levels of allergenic proteins were significantly different among the 6 germplasm entities,providing an important basis for future breeding of new poplar hypoallergenic varieties.However,further study is needed to understand the correlation between expression levels and allergenicity of the allergenic proteins of mature pollen.%[目的] 研究6份杨树种质(36号杨、南杨、110杨、森海2号杨、小叶杨和钻天杨)成熟花粉的萌发特性以及致敏蛋白差异,为低致敏杨树品种选育及植物过敏性疾病控制等研究提供理论依据.[方法] 2015 年3月,在北京、河北和河南采集6份不同杨树种质的花枝,经水培后收集花粉,采用液体培养法进行杨树花粉萌发试验,并通过双向电泳、实时荧光定量PCR等研究不同杨树种质的花粉致敏蛋白差异.[结果] 1) 在6份杨树种质花粉中,36号杨、南杨和钻天杨花粉活力较高,其次为110杨和小叶杨,森海2号杨活力最差;24 h时花粉萌发率最大的种质是36号杨,高达74.42%(±2.36%),而三倍体森海2号杨花粉萌发率为0;钻天杨花粉管平均长度为456.00 μm(±2.05 μm),小叶杨花粉管最短,为276.44 μm(±11.08 μm),且不同种质的花粉萌发率和花粉管萌发长度差异极显著(P<0.01),表明不同种质的花粉活力存在很大差异.2) 利用Imagemaster 2D platinum 6.0软件分析南杨、110杨、森海2号杨、小叶杨和钻天杨的2-DE图谱,共发现有408个蛋白点相匹配,以36号杨鉴定的28个潜在致敏蛋白为参照,在5份种质花粉中有6个致敏蛋白均缺失表达,南杨和森海2号杨花粉有21个致敏蛋白,110杨次之,有19个,而小叶杨和钻天杨的致敏蛋白数量最少,为18个.对其余22个致敏蛋白进行差异表达量分析发现,其中有12个致敏蛋白在5份杨树种质中均低于36号杨,其相对表达量范围为-3.99~-0.02,尤其是森海2号杨和小叶杨分别有10个和8个致敏蛋白表达量显著(P<0.05)或极显著(P<0.01)低于36号杨;南杨和钻天杨降低趋势最小.有3个致敏蛋白在5份种质中表达均高于36号杨,其中南杨最高,达到极显著水平(P<0.01),其次为钻天杨、森海2号杨和110杨、小叶杨.组间差异方差分析发现南杨和钻天杨的17个致敏蛋白表达量显著或极显著大于小叶杨和森海2号杨.3) qRT-PCR分析结果表明,9个致敏蛋白编码基因中有5个在南杨和钻天杨中表达量都较高,包括spots 12、51、200、216和161,其次是小叶杨,而森海2号杨和110杨均只有2个致敏基因表达高.[结论] 杨树不同种质间花粉活力、致敏蛋白数量以及表达量存在明显差异,可为今后杨树低致敏新品种选育提供重要的参考,但是成熟花粉致敏蛋白表达量的高低与其致敏性强弱的相关性还有待进一步深入研究.