5′UTR区的编辑降低大麦GBSSI基因表达

doi:10.13560/j.cnki.biotech.bull.1985.2024-0813

摘要/Abstract

摘要：

目的大麦是第四大粮食作物，其籽粒总淀粉含量以及直链淀粉和支链淀粉的比例是决定大麦品质和特性的关键因素之一，直链淀粉含量的精细调控对于大麦面粉品质改良具有重要意义。方法本研究以大麦Cas9过表达株系为受体，通过BSMV-sg介导的基因编辑系统，对GBSSI基因的5′非编码区域（5′UTR）进行靶向编辑，实现GBSSI基因表达的精细调控。结果 14株M0代5′UTR植株的第5分蘖叶片均发生了体细胞编辑，编辑效率为1.22%-84.49%，收获体细胞编辑效率最高的第5分蘖的种子，并在其23株M1代单株中鉴定到6株编辑系，编辑植株比例达26.08%。RT-qPCR检测GBSSI基因的表达，编辑系的表达量比对照下降了40%-82%。结论通过编辑5′UTR区可以调控GBSSⅠ基因的表达，为直链淀粉合成的精细调控提供新思路。

关键词: 基因编辑, BSMV-sg系统, 5′UTR, GBSSI基因, 表达调控, 大麦

Abstract:

Objective Barley is the fourth largest grain crop. The total starch content and the ratio of amylose and amylopectin in the grain are the key factors to determine the quality and characteristics of barley. The fine control of amylose content is of great significance for the improvement of barley flour quality. Method In this study, barley Cas9 overexpression lines were used as receptors, and the 5′non-coding region (5′ UTR) of GBSSI gene was targeted by BSMV-SG-mediated gene editing system to achieve the fine regulation of GBSSI gene expression. Result Our results indicated that somatic editing occurred in the leaves of the fifth tiller from 14 M0 generation 5′UTR plants, with an editing efficiency ranging from 1.22% to 84.49%. Seeds from the fifth tiller exhibiting peak editing efficiency were harvested, leading to identification of six edited lines among twenty-three M1 generation plants, resulting in an edited plant proportion of 26.08%. RT-qPCR analysis revealed a reduction in GBSSI gene expression by 40%–82% compared to controls. Conclusion These findings demonstrate that GBSSI gene expression can be effectively regulated through modification of its 5′UTR region, providing novel insights into precise regulation mechanisms for amylose synthesis.

Key words: gene editing, BSMV-sg system, 5′UTR, GBSSI gene, expression regulation, barley

薛瑞莹, 刘永菊, 姜燕燕, 彭欣雅, 曹东, 李云, 刘宝龙, 包雪梅. 5′UTR区的编辑降低大麦GBSSI基因表达[J]. 生物技术通报, 2025, 41(3): 83-89.

XUE Rui-ying, LIU Yong-ju, JIANG Yan-yan, PENG Xin-ya, CAO Dong, LI Yun, LIU Bao-long, BAO Xue-mei. Reducing the Expression of GBSSI Gene in Barley via the Editing in the 5′UTR Region[J]. Biotechnology Bulletin, 2025, 41(3): 83-89.

图/表 5

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基因名称 Gene name	引物序列 Primer sequence（5′-3′）	备注 Remark
HvUTR-cta-F	GCTGCCTCTCGCACGGTC	载体构建 Vector construction
HvUTR-aac-R	GCAAAGGAACCTCAGGGC	载体构建 Vector construction
BS8	CAACTGCCGATGATCTGTCGTGTAG	阳性克隆检测 Detection of positive clones
BS9	CCGACGCGGAAATTCGTCAAGC
BS11	GGTAGAACTGATGTGAGAGATGTAGAAG
BS32	TGGTCTTCCCTTGGGGGACCGAA
UTR-TOM-F	ggagtgagtacggtgtgcGCTGCCTCTCGCACGGTC	高通量测序 High-throughput sequencing
UTR-TOM-R	gagttggatgctggatggCGGGACTCCAAGGAAACG	高通量测序 High-throughput sequencing
WXQF1	CCAGTCCAATGGCATCTACA	RT-qPCR
WXQR1	GGCTCACCGTCAGCACCT	RT-qPCR
Tubulin-F	CAAGGAGGTGGACGAGCAGATG	RT-qPCR
Tubulin-R	GACTTGACGTTGTTGGGGATCCA	RT-qPCR

基因名称 Gene name	引物序列 Primer sequence（5′-3′）	备注 Remark
HvUTR-cta-F	GCTGCCTCTCGCACGGTC	载体构建 Vector construction
HvUTR-aac-R	GCAAAGGAACCTCAGGGC	载体构建 Vector construction
BS8	CAACTGCCGATGATCTGTCGTGTAG	阳性克隆检测 Detection of positive clones
BS9	CCGACGCGGAAATTCGTCAAGC
BS11	GGTAGAACTGATGTGAGAGATGTAGAAG
BS32	TGGTCTTCCCTTGGGGGACCGAA
UTR-TOM-F	ggagtgagtacggtgtgcGCTGCCTCTCGCACGGTC	高通量测序 High-throughput sequencing
UTR-TOM-R	gagttggatgctggatggCGGGACTCCAAGGAAACG	高通量测序 High-throughput sequencing
WXQF1	CCAGTCCAATGGCATCTACA	RT-qPCR
WXQR1	GGCTCACCGTCAGCACCT	RT-qPCR
Tubulin-F	CAAGGAGGTGGACGAGCAGATG	RT-qPCR
Tubulin-R	GACTTGACGTTGTTGGGGATCCA	RT-qPCR

Edit type	Sequence （5′-3′）	UTR-1	UTR-2	UTR-3	UTR-4	UTR-5	UTR-6	UTR-7
WT	GAGCGGAGCGGAGCGGGTATTG	7.01	25.55	98.87	96.89	19.5	98.26	98.94
+A	GAGCGGAGCGGAGCGGGTAATTG	65.26	48.83	/	/	48.72	/	/
+T	GAGCGGAGCGGAGCGGGTTATTG	13.42	7.56	/	/	11.48	/	/
+AA	GAGCGGAGCGGAGCGGGTAAATTG	1.44	/	/	/	1.96	/	/
A→G	GAGCGGAGCGGAGCGGGTGTTG	/	2.70	1.13	1.53	4.20	/	/
-GGT	GAGCGGAGCGGAGCG---ATTG	/	1.32	/	/	/	/	/
+C	GAGCGGAGCGGAGCGGGTCATTG	/	2.46	/	/	2.62	/	/
-T	GAGCGGAGCGGAGCGGG-ATTG	1.49	2.19	/	/	4.36	/	/
-AT	GAGCGGAGCGGAGCGGGT--TG	4.29	1.08	/	/	1.31	/	/
-TAT	GAGCGGAGCGGAGCGGG---TG	1.39	3.72	/	/	/	/	1.06
-GC	GAGCGGAGCGGA--GGTATTG	3.87	/	/	/	/	/	/
-GT	GAGCGGAGCGGAGCG--ATTG	/	1.62	/	/	2.26	/	/
-GGTATT	GAGCGGAGCGGAGCG------G	1.83	/	/	/	/	/	/
-ATT	GAGCGGAGCGGAGCGGGT---G	/	3.02	/	/	1.71	/	/
T→G	GAGCGGAGCGGAGCGGGGATTG	/	/	/	/	1.88	/	/
T→C	GAGCGGAGCGGAGCGGGCATTG	/	/	/	1.58	/	1.74	/
Edit type	Sequence （5′-3′）	UTR-8	UTR-9	UTR-10	UTR-11	UTR-12	UTR-13	UTR-14
WT	GAGCGGAGCGGAGCGGGTATTG	98.81	98.11	19.77	12.81	11.48	7.93	92.81
+A	GAGCGGAGCGGAGCGGGTAATTG	/	/	52.05	56.66	49.91	40.35	4.00
+T	GAGCGGAGCGGAGCGGGTTATTG	/	/	7.68	13.62	17.86	25.89	1.23
+C	GAGCGGAGCGGAGCGGGTCATTG	/	/	6.08	3.32	/	4.78	/
+AT	GAGCGGAGCGGAGCGGGTATATTG	/	/	/	4.68	/	2.47	/
+G	GAGCGGAGCGGAGCGGGGTATTG	/	/	/	3.03	6.27	5.23	/
-GT	GAGCGGAGCGGAGCGG--ATTG	/	/	1.19	/	/	/	/
-CG	GAGCGGAGCGGAG--GGTATTG	1.19	/	3.08	/	/	/	/
-TAT	GAGCGGAGCGGAGGG---TG	/	/	3.24	/	/	/	/
T→C	GAGCGGAGCGGAGCGGGCATTG	/	/	1.59	/	/	6.12	/
T→G	GAGCGGAGCGGAGCGGGGATTG	/	/	4.18	/	7.60	/	/
+GG	GAGCGGAGCGGAGCGGGTGGATTG	/	/	/	5.89	/	/	/
T→A	GAGCGGAGCGGAGCGGGAATTG	/	/	1.14	/	/	7.23	/
A→G	GAGCGGAGCGGAGCGGGTGTTG	/	1.89	/	/	6.87	/	1.96

Edit type	Sequence （5′-3′）	UTR-1	UTR-2	UTR-3	UTR-4	UTR-5	UTR-6	UTR-7
WT	GAGCGGAGCGGAGCGGGTATTG	7.01	25.55	98.87	96.89	19.5	98.26	98.94
+A	GAGCGGAGCGGAGCGGGTAATTG	65.26	48.83	/	/	48.72	/	/
+T	GAGCGGAGCGGAGCGGGTTATTG	13.42	7.56	/	/	11.48	/	/
+AA	GAGCGGAGCGGAGCGGGTAAATTG	1.44	/	/	/	1.96	/	/
A→G	GAGCGGAGCGGAGCGGGTGTTG	/	2.70	1.13	1.53	4.20	/	/
-GGT	GAGCGGAGCGGAGCG---ATTG	/	1.32	/	/	/	/	/
+C	GAGCGGAGCGGAGCGGGTCATTG	/	2.46	/	/	2.62	/	/
-T	GAGCGGAGCGGAGCGGG-ATTG	1.49	2.19	/	/	4.36	/	/
-AT	GAGCGGAGCGGAGCGGGT--TG	4.29	1.08	/	/	1.31	/	/
-TAT	GAGCGGAGCGGAGCGGG---TG	1.39	3.72	/	/	/	/	1.06
-GC	GAGCGGAGCGGA--GGTATTG	3.87	/	/	/	/	/	/
-GT	GAGCGGAGCGGAGCG--ATTG	/	1.62	/	/	2.26	/	/
-GGTATT	GAGCGGAGCGGAGCG------G	1.83	/	/	/	/	/	/
-ATT	GAGCGGAGCGGAGCGGGT---G	/	3.02	/	/	1.71	/	/
T→G	GAGCGGAGCGGAGCGGGGATTG	/	/	/	/	1.88	/	/
T→C	GAGCGGAGCGGAGCGGGCATTG	/	/	/	1.58	/	1.74	/
Edit type	Sequence （5′-3′）	UTR-8	UTR-9	UTR-10	UTR-11	UTR-12	UTR-13	UTR-14
WT	GAGCGGAGCGGAGCGGGTATTG	98.81	98.11	19.77	12.81	11.48	7.93	92.81
+A	GAGCGGAGCGGAGCGGGTAATTG	/	/	52.05	56.66	49.91	40.35	4.00
+T	GAGCGGAGCGGAGCGGGTTATTG	/	/	7.68	13.62	17.86	25.89	1.23
+C	GAGCGGAGCGGAGCGGGTCATTG	/	/	6.08	3.32	/	4.78	/
+AT	GAGCGGAGCGGAGCGGGTATATTG	/	/	/	4.68	/	2.47	/
+G	GAGCGGAGCGGAGCGGGGTATTG	/	/	/	3.03	6.27	5.23	/
-GT	GAGCGGAGCGGAGCGG--ATTG	/	/	1.19	/	/	/	/
-CG	GAGCGGAGCGGAG--GGTATTG	1.19	/	3.08	/	/	/	/
-TAT	GAGCGGAGCGGAGGG---TG	/	/	3.24	/	/	/	/
T→C	GAGCGGAGCGGAGCGGGCATTG	/	/	1.59	/	/	6.12	/
T→G	GAGCGGAGCGGAGCGGGGATTG	/	/	4.18	/	7.60	/	/
+GG	GAGCGGAGCGGAGCGGGTGGATTG	/	/	/	5.89	/	/	/
T→A	GAGCGGAGCGGAGCGGGAATTG	/	/	1.14	/	/	7.23	/
A→G	GAGCGGAGCGGAGCGGGTGTTG	/	1.89	/	/	6.87	/	1.96

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