STTM技术沉默马铃薯Stu-miR156对其侧根发育的影响

doi:10.16420/j.issn.0513-353x.2020-0230

摘要/Abstract

摘要：

为了阐明Stu-miR156在马铃薯侧根发育中的生物学功能,以马铃薯栽培品种‘Desiree’为试验材料,利用短串联靶标模拟物（Short tandem target mimic,STTM）技术,成功构建了Stu-miR156沉默表达载体,通过农杆菌介导法转化马铃薯获得了转化植株,qRT-PCR技术检测Stu-miR156及其靶基因StSPL9在转基因植株中的表达量。并通过构建pCAM-GFP-StSPL9融合蛋白表达载体转化烟草,发现靶基因StSPL9位于细胞质和细胞核中。qRT-PCR分析结果表明:在马铃薯转基因株系L1和L2中,Stu-miR156表达量受到严重抑制,在根、茎和叶中均不同程度地下调,其靶基因StSPL9均上调表达。表型分析表明:与野生型对照相比,转基因植株的侧根数量显著减少,生长受到抑制。

关键词: 马铃薯, Stu-miR156, STTM, StSPL9, 遗传转化, 侧根

Abstract:

To elucidate the biological function of Stu-miR156 in potato lateral root development,plantlets in vitro of potato cultivar‘Desiree’were used as dornors. Using STTM（Short tandem target mimic,STTM）technology,the Stu-miR156 silencing expression vector was successfully constructed and further the transgenic plants were obtained viaAgrobacterium-mediated method. qRT-PCR was used to determine the differential expression of Stu-miR156 and its target gene StSPL9in the transgenic potato plants. We constructed a pCAM-GFP-StSPL9 fusion protein expression vector and transformed it into tobacco,thereby confirming that StSPL9 was localized in the cytoplasm and nucleus. The result of qRT-PCR showed that the expression levels of Stu-miR156 in roots,stems and leaves were severely inhibited,while the target gene StSPL9 was up-regulated in the transgenic potato lines L1 and L2. The number of lateral roots of the transgenic plants was significantly reduced and the growth is inhibited compared with the wild type plants.

Key words: potato, Stu-miR156, STTM, StSPL9, genetic transformation, lateral root

中图分类号:

S532

罗红玉, 杨江伟, 冯亚, 张欢欢, 刘升燕, 张宁, 司怀军. STTM技术沉默马铃薯Stu-miR156对其侧根发育的影响[J]. 园艺学报, 2021, 48(3): 531-538.

LUO Hongyu, YANG Jiangwei, FENG Ya, ZHANG Huanhuan, LIU Shengyan, ZHANG Ning, SI Huaijun. The Effect of Stu-miR156 Silencing by STTM Technology on Potato Lateral Root Development[J]. Acta Horticulturae Sinica, 2021, 48(3): 531-538.

图/表 8

表1 核苷酸序列

Table 1 Nucleotide sequences

序列名称 Sequence name	序列（5′-3′） Sequence
Stu-miR156	UUGACAGAAGAUAGAGAGCAC
Linker（48 nt）	gttgttgttgttatggtctaatttaaatatggtctaaagaagaagaat
Stu-miR156STTM	GGATCCGTGCTCTCTCctaTCTTCTGTCAAgttgttgttgttatggtctaatttaaatatggtctaaagaagaagaatGTGCTCTCTCctaTCTTCTGTCAAAAGCTT

图1 Stu-miR156靶基因预测及靶基因结构分析

Fig. 1 Prediction of Stu-miR156 target gene and analysis of target gene structure

图2 pCAM-GFP-SPL9蛋白的亚细胞定位

Fig. 2 Subcellular localization of the pCAM-GFP-SPL9 protein

图3 Stu-miR156STTM载体（A）与重组表达载体pCAMBIA1301-Stu-miR156STTM（B）结构示意图

Fig. 3 Schematic diagram of Stu-miR156STTM vector（A）and recombinant expression vector pCAMBIA1301-Stu-miR156STTM（B）

图4 Stu-miR156STTM转基因植株PCR鉴定

Fig. 4 PCR identification of Stu-miR156STTM transgenic plants

图5 Stu-miR156STTM 转基因植株（L1和L2）相对表达量分析不同小写字母表示差异显著（P < 0.05）。

Fig. 5 Analysis of relative expression of the transgenic plants（L1 and L2）with Stu-miR156STTM transformation Different lowercase letters indicate significant differences（P < 0.05）.

表2 马铃薯转基因植株L1和L2的表型测定

Table 2 Phenotypic assay of the transgenic potato plants L1 and L2

株系 Plant line	株高/mm Height	根数 Root number	根长/mm Root length	根冠比/% Root-shoot ratio
野生型Wild type	72.08 ± 5.05 a	33.33 ± 3.68 a	27.33 ± 1.25 a	38.03 ± 2.03 a
L1	69.42 ± 3.37 a	5.67 ± 2.05 b	26.33 ± 1.70 ab	38.02 ± 3.09 a
L2	65.02 ± 6.51 a	8.33 ± 1.70 b	22.67 ± 1.70 b	34.97 ± 0.84 a

图6 Stu-miR156STTM 转基因马铃薯植株表型

Fig. 6 Phenotypes of the transgenic potatoes with Stu-miR156STTM transformation

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