摘要
本文选取不同整合方式的转基因白桦无性系,以其微繁过程中不同发育阶段(腋芽、幼嫩愈伤组织、老化愈伤组织、出芽愈伤、再生芽、根)及特殊状态愈伤组织(花药愈伤组织、红斑状愈伤组织、木质化愈伤)作为研究材料。测定其POD、SOD、CAT、木质素、可溶性糖、可溶性蛋白、丙二醛等生理生化指标。利用甲基化敏感多态性(methylation-sensitive amplified polymorphism, MSAP)检测微繁过程中DNA甲基化水平和模式的变化。以酶联免疫技术(ELISA)测定甲基转移酶活性的变化,应用荧光定量PCR技术测定甲基转移酶相关基因(DRM和MET, DRM是重头甲基化酶(de novo methyltransferases)的基因,MET是维持甲基化酶(maintenance methyltransferase)的基因)及外源基因(BGT)的表达水平。从而揭示微繁再生过程中的DNA甲基化变异机制。所得结果如下：
1.微繁过程中(脱分化和再分化),POD、SOD和CAT三个保护酶活性、可溶性糖含量、呈先升高(1～2倍)后降低(0.3～0.7倍)趋势,而木质素和可溶性糖呈先降低后升高趋势。老化愈伤组织保护酶活性、可溶性糖降低仅为幼嫩愈伤组织的0.2～0.4倍。愈伤组织老化阶段保护酶活性、木质素和可溶性总蛋白含量显著降低(0.3～0.7倍),MDA含量升高(1.5倍)。
2.DNA甲基化水平分析表明,微繁过程中不同阶段的转基因Tp46号株系为9.93%～14.77%,转基因Tp74号株系为13.33%～21.22%,非转基因对照(CK)为11.92%～17.03%,3个无性系变化趋势相似,在微繁过程中(脱分化和再分化),呈先降低后升高的趋势,平均值由13.62%下降至11.89%,又上升至13.42%,腋芽诱导形成愈伤组织阶段主要发生去甲基化,其MSAP模式变化以出现新条带为主(64.52%～92.59%)。再生芽诱导生根阶段,DNA甲基化平均值由14.70%升高到16.79%, MSAP模式以条带消失为主(79%)。老化愈伤组织DNA甲基化平均值(15.06%)高于幼嫩愈伤组织(11.89%),其MSAP模式变化以条带消失为主(55.56%)。红斑状愈伤组织和木质化愈伤组织甲基化水平略高于幼嫩愈伤组织。MSAP模式中内外侧甲基化位点转换频率最低,幼嫩愈伤组织形成再生芽过程最高仅为6.85%。
3.甲基转移酶基因表达,微繁过程(脱分化和再分化),除Tp46号MET基因表达含量升高外,3个无性系两个甲基转移酶基因表达都降低,最低仅为腋芽的0.06倍,愈伤组织形成再生芽过程中,甲基转移酶基因表达水平升高,最高为腋芽的6.73倍。特殊状态愈伤组织甲基转移酶活性均高于幼嫩愈伤组织,最高老化愈伤组织为幼嫩愈伤的20倍。
4.外源基因表达,在微繁过程(脱分化和再分化)外源基因表达先升高后降低,升高倍数约为1.2倍,生根过程外源基因表达显著降低,根中仅为腋芽的0.36倍。老化愈伤组织、花药愈伤组织的外源基因表达水平低于幼嫩愈伤,约为幼嫩愈伤组织的0.6倍。
In this study, We selected different transgenic birch clones, including different stages of micropropagation process(buds, young callus, aging callus, sprouting callus, buds, roots)and the special status of callus(anther callus, erythema-like callus, lignified callus, the red callus). The POD activity, SOD activity, CAT activity, lignin content, soluble sugar content, soluble protein content, MDA content were measured. DNA methylation level and patterns were revealed by MSAP. The methyltransferase activity changes and methyltransferase gene(DRM and MET) and the foreign gene(BGT) changes in expression levels were analyzed by ELISA and real-time PCR. The results were as follows:
1. In the process of micropropagation(dedifferentiation and regeneration), the activity of POD, SOD and CAT and soluble sugar were first increased(1 to 2 times than control) and then decreased(0.3 to 0.7 times than control). The content of lignin and soluble sugar were opposite. The POD, SOD, CAT activity, lignin and soluble protein content in aging callus were significantly decreased, but the MDA content was increased(1.5 times).
2. The DNA methylation level of Tp46 wes from 9.93% to 14.77%, the Tp74 was from 13.33% to 21.22%, CK was from 11.92% to 17.03% in the micropropagation process(dedifferentiation and regeneration). The average DNA methylation level reduced from 13.62% to 11.89% first, then increase to 13.42%. The phase of buds induced into callus mainly demethylatied, whereas the pattern of MSAP is increasing bands (64.52%-92.59%). The average of DNA methylation in rooting stage were increased from 14.70% to 16.79%, while the main MSAP pattern was decreasing bands(55.56%). The methylation level of red callus and lignified callus were higher than young callus. The conversion of insite and outsite methylation was the lowers, the young callus indeced into regenerated bud only up to 6.85%.
3. DRM is the gene of de novo methyltransferases, while, MET is the gene of maintenance methyltransferase. During the micropropagation process, the phase of bud induced into callus, the expression levels of MET and DRM were increased except MET of Tp46. The methyltransferase activiti of special status callus were higher than the young callus, the highest was the aging callus, about 20 times to young callus.
4. The expression of exogenous gene BGT during the micropropagation (dedifferentiation and regeneration) was first increased(1.2 times), then decreased. During rooting stage, the expression of BGT was significantly reduced only to 0.36 times than control. The special status calluses were lower than the young callus (0.6 times).
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