利用指纹图谱技术鉴定新疆油葵杂交种纯度的研究
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摘要
本文以新疆推广种植的油用向日葵品种（康地4、5、6、7的父母本及杂交种，G101的父母本及杂交种）为材料，利用蛋白质电泳技术和RAPD分子标记技术对供试油葵杂种进行纯度鉴定的研究。
应用蛋白质电泳技术的研究结果表明：（1）适宜的同工酶和种子蛋白凝胶浓度及交联度能提高电泳结果的清晰度和分辨率；（2）通过几种提取剂的比较，确定用1mol/L尿素作为种子蛋白提取剂，此提取剂提取的蛋白丰富，但差异性较小；（3）利用酯酶和过氧化物酶对供试品种进行纯度鉴定，康地6号杂交种的酯酶谱带与其父母本的特征谱带互补，其纯度鉴定结果与田间检测结果基本一致；G101杂交种的过氧化物酶谱带中出现新谱带，用此谱带鉴定G101杂交种纯度的结果也与田间鉴定结果基本一致。
应用RAPD分子标记技术的研究结果表明：（1）用不同提取方法（CTAB法、SDS法）及不同材料（真叶、种子）进行DNA的提取，确定了利用SDS法从向日葵种子中快速、简捷提取总DNA的方法；（2）对RAPD技术中的反应因子（模板浓度、Taq酶浓度、引物浓度、dNTPs浓度等）及反应条件（退火温度，延伸时间等）进行优化，确定了适用于油用向日葵RAPD分析的反应体系；（3）用100个引物对供试品种进行筛选，筛选结果是：引物OPB05在康地6号、康地7号的杂交种中扩增出各自双亲的特征谱带；引物OPB03扩增出康地4号杂交种的谱带既包括来自父本的特征谱带也包括来自母本的特征谱带；康地6号杂交种利用引物OPC14也扩增出了父母本互补的特征谱带（4）利用筛选出的引物对三个品种的杂交种进行纯度鉴定，鉴定结果与田间鉴定结果接近。
In this thesis, we conducted research work in identification of purity of oil sunflower (H. annuus) hybrids grown in Xinjiang with protein electrophoresis technique and RAPD molecular markers. We used several oil sunflower varieties --- Kangdi No.4, No.5, No.6, No.7 and G101 (including hybrid and its parents) as experimental materials, results were shown as following.
Results of protein electrophoresis showed: (1) proper concentration and crosslinking degree of gel could improve the resolution of the electrophoresis bands; (2) comparing results of SDS-PAGE of seed protein with various protein extraction solutions, we chose the one which included 1mol/L urea in it as the extraction solution, its protein was rich and electrophoresis bands appeared better, but the special and characteristic band was few; (3) results of electrophoresis of the esterase and peroxidase isozymes in cotyledon stage showed: in Kangdi No.6 hybrid, there were some special bands of esterase isozyme coming from its parents separately, that is, the hybrid had parental complementary characteristic bands. With this result, production hybrids were tested and the results were identical with that of field planting identification; in G101 hybrid, there was one new band of peroxidase isozyme in cotyledon stage which didn't appear in result of parents electrophoresis bands. Result of purity testing to G101 production hybrid with this special band also matched well with that of field planting identification.
Results of RAPD-PCR showed: (1) comparing different methods (CTAB, SDS) of total DNA extraction from different materials (young leaves, seed), we considered that SDS method using seed as material was much simpler and quicker than others; (2) In optimizing of the reaction components (concentration of template DNA, Taq DNA polymerase, primer, dNTPs etc) and procedure (annealing temperature, extention time, etc.) of RAPD-PCR, we developed the optimization reaction systemof RAPD-PCR for oil sunflower in our lab; (3) using 100 random 10-mer oligonucleotide primers screened varieties mentioned above with the optimization reaction system of RAPD-PCR, results showed that primer OPB05 generated the different parental complementary characteristic bands in Kangdi No.6, No.7 hybrids separately; Kangdi No.4 hybrids with primer OPB03, Kangdi No.6 hybrid with primer OPC14 could also generate parental complementary special bands; (4) with above results, three hybrids (Kangdi No.4, No.6, No.7) of their production seeds were tested, and the results all matched well with those of field planting identification.
应用蛋白质电泳技术的研究结果表明：（1）适宜的同工酶和种子蛋白凝胶浓度及交联度能提高电泳结果的清晰度和分辨率；（2）通过几种提取剂的比较，确定用1mol/L尿素作为种子蛋白提取剂，此提取剂提取的蛋白丰富，但差异性较小；（3）利用酯酶和过氧化物酶对供试品种进行纯度鉴定，康地6号杂交种的酯酶谱带与其父母本的特征谱带互补，其纯度鉴定结果与田间检测结果基本一致；G101杂交种的过氧化物酶谱带中出现新谱带，用此谱带鉴定G101杂交种纯度的结果也与田间鉴定结果基本一致。
应用RAPD分子标记技术的研究结果表明：（1）用不同提取方法（CTAB法、SDS法）及不同材料（真叶、种子）进行DNA的提取，确定了利用SDS法从向日葵种子中快速、简捷提取总DNA的方法；（2）对RAPD技术中的反应因子（模板浓度、Taq酶浓度、引物浓度、dNTPs浓度等）及反应条件（退火温度，延伸时间等）进行优化，确定了适用于油用向日葵RAPD分析的反应体系；（3）用100个引物对供试品种进行筛选，筛选结果是：引物OPB05在康地6号、康地7号的杂交种中扩增出各自双亲的特征谱带；引物OPB03扩增出康地4号杂交种的谱带既包括来自父本的特征谱带也包括来自母本的特征谱带；康地6号杂交种利用引物OPC14也扩增出了父母本互补的特征谱带（4）利用筛选出的引物对三个品种的杂交种进行纯度鉴定，鉴定结果与田间鉴定结果接近。
In this thesis, we conducted research work in identification of purity of oil sunflower (H. annuus) hybrids grown in Xinjiang with protein electrophoresis technique and RAPD molecular markers. We used several oil sunflower varieties --- Kangdi No.4, No.5, No.6, No.7 and G101 (including hybrid and its parents) as experimental materials, results were shown as following.
Results of protein electrophoresis showed: (1) proper concentration and crosslinking degree of gel could improve the resolution of the electrophoresis bands; (2) comparing results of SDS-PAGE of seed protein with various protein extraction solutions, we chose the one which included 1mol/L urea in it as the extraction solution, its protein was rich and electrophoresis bands appeared better, but the special and characteristic band was few; (3) results of electrophoresis of the esterase and peroxidase isozymes in cotyledon stage showed: in Kangdi No.6 hybrid, there were some special bands of esterase isozyme coming from its parents separately, that is, the hybrid had parental complementary characteristic bands. With this result, production hybrids were tested and the results were identical with that of field planting identification; in G101 hybrid, there was one new band of peroxidase isozyme in cotyledon stage which didn't appear in result of parents electrophoresis bands. Result of purity testing to G101 production hybrid with this special band also matched well with that of field planting identification.
Results of RAPD-PCR showed: (1) comparing different methods (CTAB, SDS) of total DNA extraction from different materials (young leaves, seed), we considered that SDS method using seed as material was much simpler and quicker than others; (2) In optimizing of the reaction components (concentration of template DNA, Taq DNA polymerase, primer, dNTPs etc) and procedure (annealing temperature, extention time, etc.) of RAPD-PCR, we developed the optimization reaction system
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106
107 Hu ZA(胡志昂), Wang HX(王洪新). Protein diversity and cultivar identification. Acta Bot Sinca(植物学报). 1991, 33 (7):556~564
108 颜启传，邓光联，支巨振，等主编。农作物品种电泳鉴定手册。上海：上海科学技术出版社，1998，p77.
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