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作物学报 ›› 2019, Vol. 45 ›› Issue (5): 683-692.doi: 10.3724/SP.J.1006.2019.84118

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

基于Cre/loxP系统的无筛选标记转耐低磷转录因子GmPTF1大豆种质创制与分析

张小芳1,董秋平1,乔潇2,乔亚科1,*(),王冰冰1,张锴1,李桂兰1,*()   

  1. 1 河北科技师范学院农学与生物科技学院, 河北昌黎 066600
    2 河北科技师范学院物理系, 河北秦皇岛066004
  • 收稿日期:2018-08-27 接受日期:2018-12-24 出版日期:2019-05-12 网络出版日期:2019-01-16
  • 通讯作者: 乔亚科,李桂兰
  • 基金资助:
    本研究由国家转基因生物新品种培育科技重大专项(2014ZX0800404B);现代农业产业技术体系河北省创新团队建设项目(HBCT2018090203)

Creation and analysis of marker free transgenic soybean germplasm with low phosphate tolerance transcription factor GmPTF1 based on Cre/loxP system

Xiao-Fang ZHANG1,Qiu-Ping DONG1,Xiao QIAO2,Ya-Ke QIAO1,*(),Bing-Bing WANG1,Kai ZHANG1,Gui-Lan LI1,*()   

  1. 1 College of Agronomy and Biotechnology, Hebei Normal University of Science and Technology, Changli 066600, Hebei, China
    2 Department of Physics, Hebei Normal University of Science and Technology, Qinhuangdao 066004, Hebei, China
  • Received:2018-08-27 Accepted:2018-12-24 Published:2019-05-12 Published online:2019-01-16
  • Contact: Ya-Ke QIAO,Gui-Lan LI
  • Supported by:
    This study was supported by the National Transgenic Major Project of China(2014ZX0800404B);Modern Agricultural Industrial Technology System Hebei Province Innovation Team Construction Project(HBCT2018090203)

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摘要：

筛选标记基因在转基因植物应用中存在一定潜在安全风险, 在转基因植物改良中如何合理消除该基因非常必要。转录因子PTF1具有改善植物在低磷胁迫下吸收磷效率的作用。本研究用根癌农杆菌介导法将Cre/loxP- GmPTF1导入大豆品种豫豆22, 利用β-雌二醇诱导Cre/loxP系统删除筛选标记基因, 获得了无筛选标记的转GmPTF1基因大豆。用PCR法扩增删除标记基因后的重组序列并测序显示, 筛选标记基因在大豆基因组中已经被完全删除, 重组序列中目的基因序列正确并保持了正确开放读码框; loxP位点重组出现一种新的拼接类型, 重组后2个loxP序列全部缺失, 新重组拼接位点长38 bp, 与NCBI数据库的其他序列均无同源性, 并且重组涉及2个loxP位点的外侧翼序列, 造成Cre/loxP 盒上游loxP和下游loxP外侧翼序列部分缺失。经过RT-PCR和Western杂交验证显示无筛选标记转基因大豆植株中GmPTF1能够正常转录和翻译, 在根系、叶片及茎中的GmPTF1蛋白表达量均高于野生型对照, 而在种子中与对照无显著差异。沙培试验表明, 在低磷条件下无筛选标记转基因大豆苗期根系指标、生物干重、叶绿素含量和磷含量均显著高于野生型对照, 而丙二醛含量低于对照。利用Cre/loxP重组系统可以有效删除转基因大豆中的筛选标记基因。

关键词: Cre/loxP, GmPTF1, 无筛选标记, 转基因大豆

Abstract:

The screening marker genes in transgenic crops have potential safety risks, which are necessarily eliminated in the improvement of transgenic plants. The transcription factor PTF1 has the effect improving phosphorus uptake by plants under low phosphorus stress. So Cre/loxP-GmPTF1 was transferred into soybean cultivar Yudou 22 [Glycine max (L.) Merr.] via Agrobacterium-mediated cotyledonary node transformation method. And then, the marker-free transgenic soybean with GmPTF1 was obtained by means of induced Cre/loxP site specific recombination with β-estradiol. After the marker gene was deleted, the recombinant sequences were amplified by PCR and then sequenced. The sequencing results indicate that the marker genes were completely deleted out of the transgenic soybean genome. The recombinant maintained with correct ORF correct target gene sequence, and a new splicing type appeared in loxP recombination. Two loxP sequences were missing in the recombination. The length of the new recombinant in splicing site was 38 bp which was no homology with other sequences in NCBI database. And the recombination was involved in the flank sequence outside the two loxP sites, which lead partially deleted in the outer flank sequences of Cre/loxP cassette. The results of RT-PCR and Western blot showed that GmPTF1 could be transcribed and translated normally in marker-free transgenic soybean plants. The expression level of GmPTF1 in roots, stem and leaves was higher than that in wild type, which was not significantly different in seeds. in the sand culture experiment under low phosphorus condition, the root indexes, dry biomass, chlorophyll content and phosphorus content in marker-free transgenic soybean were significantly higher than there in the wild type, while the MDA content was lower than that in the control. We conclude that screening marker genes in transgenic soybean could be effectively deleted by Cre/loxP recombinant system.

Key words: Cre/loxP, GmPTF1, marker-free, transgenic soybean

图1

标记基因删除前(A)和删除后(B)转基因植株插入序列及PCR鉴定示意图"

图2

转基因植株删除标记之前的PCR检测图 1: 100 bp DNA ladder marker; 2: 水对照; 3: 野生型对照; 4, 5: P1/P2 PCR产物; 6, 7: P3/P4 nptII PCR产物; 8, 9: P5/P6 GmPTF1基因PCR产物。"

图3

转基因植株删除标记之后的PCR检测图 1, 12: 100 bp DNA ladder marker; 2: 水对照; 3: 野生型对照; 4, 5: P1/P2载体骨架片段PCR产物; 6, 7: P3/P4 nptII PCR产物; 8, 9: P5/P6 GmPTF1基因PCR产物; 10, 11: P1/P6 PCR产物, 删除标记后重组DNA片段。"

图4

标记基因删除后重组序列P1/P6 PCR产物测序结果分析 阴影部分为P1和P6引物序列, 线框内为重组拼接位点。"

图5

无筛选标记转GmPTF1基因大豆T5代Southern blot检测图 1: 转基因材料Xba I酶切Southern blot; 2: 转基因材料Kpn I酶切Southern blot; WT: 野生型对照Xba I酶切Southern blot; M: λDNA/Hind III marker。"

图6

无筛选标记转基因大豆GmPTF1基因RT-PCR H: 空白对照; WT: 野生型对照; 1~7: 转基因植株; P: 质粒阳性对照; M: 100 bp ladder DNA marker。"

图7

无筛选标记转基因大豆GmPTF1转录水平分析 1: 野生型; 2: 转基因大豆。"

图8

无筛选标记转基因大豆不同器官中GmPTF1蛋白的Western杂交结果 1: 野生型对照; 2: 转基因材料; A: GmPTF基因表达蛋白; B: Actin基因表达蛋白。"

图9

无筛选标记转基因大豆不同器官中GmPTF1蛋白相对表达量 1: 野生型对照; 2: 转基因大豆; A: 根; B: 叶; C: 茎; D: 种子。"

表1

不同磷浓度下不同大豆植株的根系性状"

材料
Material		 根总长
Root length (cm)		 根表面积
Root surface area (cm2)		 根平均直径
Root average diameter (mm)		 根体积
Root volume (cm3)
正常磷
Normal-P		 低磷
Low-P		 正常磷
Normal-P		 低磷
Low-P		 正常磷
Normal-P		 低磷
Low-P		 正常磷
Normal-P		 低磷
Low-P
Transgenic line 2515±99 aA 2175±49 aA 146±4.8 aA 113±4.1 aA 0.58±0.04 aA 0.52±0.01 aA 6.67±0.71 aA 4.48±0.29 aA
WT 2269±14 bA 1632±29 bB 147±6.5 aA 85±2.2 bB 0.56±0.06 aA 0.48±0.01 bB 6.25±0.95 aA 3.51±0.15 bB

表2

低磷胁迫对不同大豆幼苗地上部的影响"

材料
Material		 株高 Plant height (cm) 第一展开叶片面积 The first developed blade area (cm2)
正常磷
Normal-P		 低磷
Low-P		 降幅
Drop (%)		 正常磷
Normal-P		 低磷
Normal-P		 降幅
Drop (%)
Transgenic line 30.3±0.6 aA 24.0±1.2 Aa 20.0 20.28±0.67 aA 19.69±1.51 aA 2.96
WT 37.7±1.5 aA 23.8±2.3 aA 36.9 21.61±1.86 aA 16.06±1.25 bA 25.7

表3

低磷对不同转基因大豆干物质积累的影响"

材料
Material		 根干重 Dry weights of roots 茎叶干重 Dry weights of stems and leaves
正常磷
Normal-P		 低磷
Low-P		 降幅
Drop (%)		 正常磷
Normal-P		 低磷
Normal-P		 降幅
Drop (%)
Transgenic line 0.88±0.13 aA 0.61±0.04 aA 30.7 2.81±0.1 aA 1.38±0.06 aA 51
WT 0.85±0.04 aA 0.38±0.06 bB 55.3 2.6±0.25 aA 0.85±0.05 bB 68

图10

大豆叶片中光合色素与丙二醛含量"

表4

大豆叶片丙二醛与光合色素变幅"

材料
Material		 光合色素降幅
Drop of photosynthetic pigments (%)		 丙二醛增幅
Increasing range of MDA (%)
Transgenic line 20.81 ± 0.03 bB 29.43 ± 3.13 bB
WT 49.76 ± 0.02 aA 64.57 ± 2.09 aA

图11

不同磷浓度处理下大豆叶片磷含量 1: 野生型对照; 2: 转基因大豆。"

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