基因枪法将HMW-GS（5+10）基因转入小麦的研究
摘要
本试验采用基因枪方法，以带有小麦高分子量麦谷蛋白5亚基和10亚基基因的高效表达载体转化小麦受体品种豫麦18号、豫麦21号、豫麦49号和豫农9901，以期得到高效表达高分子量麦谷蛋白5亚基和10亚基的小麦新种质。为了减少转基因沉默、提高外源基因表达量，在目的基因的两侧构建了核基质结合序列MAR，并在基因末端加上了内质网滞留信号（KDEL）编码序列以期提高目的基因表达水平。
对基因枪轰击小麦幼胚愈伤组织过程中几个重要参数的研究结果表明，在同等条件下250 μg 枪-1的金粉用量优于500 μg 枪-1，颗粒直径1.0 μm的金粉转化效果好于直径1.6 μm金粉，单枪与双枪差别不大，可裂膜片压力为1300 psi时，轰击后愈伤组织的再生绿点率明显高于可裂膜片为1100 psi时的绿点率，9 cm靶距的处理效果较好。
试验中4个基因型共得到146株再生植株，对5亚基基因进行的PCR鉴定表明在所有146株植株中，出现预期大小DNA片段的植株有141株，有5株未扩增出预期片段，平均PCR阳性率达到96.57%。其中豫麦18号117株中有114株PCR检测为阳性，阳性率达到97.44%，豫麦21号3株为阳性，豫麦49号24株中22株检测结果为阳性，阳性率为91.67%，豫农9901仅有1株，PCR检测结果为阳性。对10亚基基因进行的PCR鉴定表明出现预期大小DNA片段的植株有138株，有8株未扩增出预期片段，平均PCR阳性率达到94.52%。其中豫麦18号117株中有112株PCR检测为阳性，阳性率达到95.72%，豫麦21号4株均为阳性，豫麦49号24株中21株检测结果为阳性，阳性率为87.5%，豫农9901 1株，PCR检测结果为阳性。
对转基因植株T0代的种籽所做SDS-PAGE的电泳结果表明，豫麦21号、豫麦49号、豫农9901的种籽高分子量麦谷蛋白亚基SDS-PAGE带型均未发生变化，豫麦18号117株中，有7株的种籽蛋白带型发生了变化，与未转基因的对照相比，这7株的高分子量麦谷蛋白带型均出现比对照的带型多出2条比较明显的带。但是，这2条带的位置与预期的5亚基与10亚基在SDS-PAGE上的位置并不一致。7株带型有变化的再生植株全部为不带KDEL编码序列质粒的转化植株，这一现象有待进一步研究。
To get high molecular weight glutenin subunit 5 and subunit 10 overexpressed wheat lines, wheat cultivar, Yumai 18, Yumai 21, Yumai 49 and Yunong 9901,were transformed with wheat high molecular weight glutenin subunit 5 and subunit 10 genes high-efficient expression vector by means of biolistic bombardment. In order to reduce transgene silencing and enhance expression of foreign gene, the two target genes were flanked with nuclear matrix attachment region, the MAR sequence. And coding sequence of KDEL, ER retention signal, was added to the end of the target genes to enhance the expression of target genes.
To improve transformation of wheat immature embryo calli, the influence of several key parameters in microprojectile bombardment were studied. The results showed that under identical conditions, transformation ratio of Au dosage of 250 μg per shot was higher than that of 500 μg per shot and Au particle of 1. 0 μm in diameter was better than that of 1. 6 μm. And there was no significant difference between single shot and double shots. When the rupture pressure was 1300 psi, percentage of regenerated green spots after bombardment was much higher than that of 1100 psi. In target distance (6, 9 and 12 cm) experiment, effect of 9 cm was the best.
146 regenerated plantlings were gotten from the 4 genotypes. Among the 146 plantlings, PCR identification of subunit 5 gene indicated that 141 plantlings showed expected DNA band, but 5 plantlings didn’t. The average PCR positive ratio was 96. 57%. Among 117 plantlings of Yumai 18, 114 plantlings were PCR positive, and the positive ratio was 97. 44%. 3 plantlings of Yumai 21 was positive, and 22 plantlings of 24 Yumai 49 plantlings were positive, the positive ratio was 91. 67%. There was only 1 plantling of Yunong 9901, and the PCR detection result was positive. PCR identification of subunit 10 gene indicated that 138 plantlings showed expected DNA band, and 8 plantlings didn’t. The average PCR positive ratio was 94. 52%. Among 117 plantlings of Yumai 18, 112 plantlings were PCR positive, and the positive ratio was 95. 72%. All the 3 plantlings of Yumai 21 were positive, and 21 plantlings of 24 Yumai 49 plantlings were positive, the positive ratio was 87. 5%. The only plantling of Yunong 9901 was positive.
In SDS-PAGE experiment of the seeds, the HMW subunit SDS-PAGE band patterns of Yumai 21, Yumai 49 and Yunong 9901 didn’t show any apparent change. Among the 117 regenerated plants of Yumai 18, 7 plantlings showed seed HMW-glutenin SDS-PAGE band pattern change. Compared to wild type, there were 2 extra HMW-glutenin bands. But the position of the 2 bands was not consistent with the expected position of subunit 5 and subunit 10. And moreover, the 7 regenerated plants with changed band pattern were all in the transformants of plasmid without KDEL coding sequence. This interesting result needs to be researched further.
对基因枪轰击小麦幼胚愈伤组织过程中几个重要参数的研究结果表明，在同等条件下250 μg 枪-1的金粉用量优于500 μg 枪-1，颗粒直径1.0 μm的金粉转化效果好于直径1.6 μm金粉，单枪与双枪差别不大，可裂膜片压力为1300 psi时，轰击后愈伤组织的再生绿点率明显高于可裂膜片为1100 psi时的绿点率，9 cm靶距的处理效果较好。
试验中4个基因型共得到146株再生植株，对5亚基基因进行的PCR鉴定表明在所有146株植株中，出现预期大小DNA片段的植株有141株，有5株未扩增出预期片段，平均PCR阳性率达到96.57%。其中豫麦18号117株中有114株PCR检测为阳性，阳性率达到97.44%，豫麦21号3株为阳性，豫麦49号24株中22株检测结果为阳性，阳性率为91.67%，豫农9901仅有1株，PCR检测结果为阳性。对10亚基基因进行的PCR鉴定表明出现预期大小DNA片段的植株有138株，有8株未扩增出预期片段，平均PCR阳性率达到94.52%。其中豫麦18号117株中有112株PCR检测为阳性，阳性率达到95.72%，豫麦21号4株均为阳性，豫麦49号24株中21株检测结果为阳性，阳性率为87.5%，豫农9901 1株，PCR检测结果为阳性。
对转基因植株T0代的种籽所做SDS-PAGE的电泳结果表明，豫麦21号、豫麦49号、豫农9901的种籽高分子量麦谷蛋白亚基SDS-PAGE带型均未发生变化，豫麦18号117株中，有7株的种籽蛋白带型发生了变化，与未转基因的对照相比，这7株的高分子量麦谷蛋白带型均出现比对照的带型多出2条比较明显的带。但是，这2条带的位置与预期的5亚基与10亚基在SDS-PAGE上的位置并不一致。7株带型有变化的再生植株全部为不带KDEL编码序列质粒的转化植株，这一现象有待进一步研究。
To get high molecular weight glutenin subunit 5 and subunit 10 overexpressed wheat lines, wheat cultivar, Yumai 18, Yumai 21, Yumai 49 and Yunong 9901,were transformed with wheat high molecular weight glutenin subunit 5 and subunit 10 genes high-efficient expression vector by means of biolistic bombardment. In order to reduce transgene silencing and enhance expression of foreign gene, the two target genes were flanked with nuclear matrix attachment region, the MAR sequence. And coding sequence of KDEL, ER retention signal, was added to the end of the target genes to enhance the expression of target genes.
To improve transformation of wheat immature embryo calli, the influence of several key parameters in microprojectile bombardment were studied. The results showed that under identical conditions, transformation ratio of Au dosage of 250 μg per shot was higher than that of 500 μg per shot and Au particle of 1. 0 μm in diameter was better than that of 1. 6 μm. And there was no significant difference between single shot and double shots. When the rupture pressure was 1300 psi, percentage of regenerated green spots after bombardment was much higher than that of 1100 psi. In target distance (6, 9 and 12 cm) experiment, effect of 9 cm was the best.
146 regenerated plantlings were gotten from the 4 genotypes. Among the 146 plantlings, PCR identification of subunit 5 gene indicated that 141 plantlings showed expected DNA band, but 5 plantlings didn’t. The average PCR positive ratio was 96. 57%. Among 117 plantlings of Yumai 18, 114 plantlings were PCR positive, and the positive ratio was 97. 44%. 3 plantlings of Yumai 21 was positive, and 22 plantlings of 24 Yumai 49 plantlings were positive, the positive ratio was 91. 67%. There was only 1 plantling of Yunong 9901, and the PCR detection result was positive. PCR identification of subunit 10 gene indicated that 138 plantlings showed expected DNA band, and 8 plantlings didn’t. The average PCR positive ratio was 94. 52%. Among 117 plantlings of Yumai 18, 112 plantlings were PCR positive, and the positive ratio was 95. 72%. All the 3 plantlings of Yumai 21 were positive, and 21 plantlings of 24 Yumai 49 plantlings were positive, the positive ratio was 87. 5%. The only plantling of Yunong 9901 was positive.
In SDS-PAGE experiment of the seeds, the HMW subunit SDS-PAGE band patterns of Yumai 21, Yumai 49 and Yunong 9901 didn’t show any apparent change. Among the 117 regenerated plants of Yumai 18, 7 plantlings showed seed HMW-glutenin SDS-PAGE band pattern change. Compared to wild type, there were 2 extra HMW-glutenin bands. But the position of the 2 bands was not consistent with the expected position of subunit 5 and subunit 10. And moreover, the 7 regenerated plants with changed band pattern were all in the transformants of plasmid without KDEL coding sequence. This interesting result needs to be researched further.
引文
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