叶面喷施TaSBEIIbs-dsRNA提高小麦直链淀粉含量

doi:10.3864/j.issn.0578-1752.2025.22.001

摘要/Abstract

摘要：

【目的】小麦淀粉主要由直链淀粉和支链淀粉构成，长期食用精加工的精制面粉制品，会增加患糖尿病等慢性病的风险，而食用抗性淀粉含量高的面粉则对调节人体血糖水平具有积极作用，鉴于抗性淀粉和直链淀粉之间一般呈正相关关系，因此，提高小麦种质中直链淀粉的含量为品质改良育种研究奠定基础。【方法】选取淀粉分支酶（starch branch enzyme）TaSBEIIb中4个SBE基因片段，成功构建TaSBEIIb-dsRNA高效表达载体，并基于培养基组分进行梯度优化，显著提升dsRNA的产量。基于此，通过叶面喷施，探究裸露dsRNA及纳米载体壳聚糖季铵盐（hydroxypropyltrimethyl ammonium chitosan chloride，HACC）包被的dsRNA对小麦淀粉代谢的调控效应；借助小麦幼苗培养系统，观察dsRNA喷洒后对小麦幼苗中直链淀粉含量的影响，以及淀粉相关基因的表达情况；进一步借助大田试验，分析dsRNA喷雾对小麦成熟籽粒中直链淀粉含量的影响；利用壳聚糖季铵盐包被的dsRNA，探究包材对dsRNA保护和对小麦成熟籽粒中直链淀粉含量的影响。【结果】成功构建了4个表达dsRNA的重组质粒pRNAi-TaSBE1—pRNAi-TaSBE4；优化后的发酵培养基使dsRNA的产量从26.54 mg·L^-1提高至50.65 mg·L^-1，相较于初始培养基提高了91%；dsRNA喷雾后干扰了目标基因的表达，其中，TaSBEIIb1片段在第7天的干扰效率最高，4个片段干扰后，TaSBEIIb的表达量平均降低47.73%；同时，TaSBEIIb的干扰也影响了淀粉合成通路中其他基因的表达，包括TaSSII、TaSSIV和TaSBEIIa1，分别在第3、7和3天影响效率最高，其表达水平分别平均降低54.53%、59.94%和47.64%；2023年田间试验表明，裸dsRNA喷洒使小麦籽粒直链淀粉含量在处理后7 d提升17.2%—36.5%，但在小麦成熟期效果衰减至0.2%—8.3%；2024年田间试验中，多次喷施裸dsRNA和壳聚糖季铵盐包被的dsRNA均能使小麦成熟籽粒中直链淀粉含量从27.72%提高至30.37%，相较于对照提升约10%。【结论】外源喷洒TaSBEIIbs-dsRNA具有提升淀粉中直链淀粉含量的作用。

关键词: 小麦, dsRNA, 淀粉分支酶, 直链淀粉, 品质改良

Abstract:

【Objective】Wheat starch mainly consists of amylose and amylopectin. Long-term consumption of refined flour products increases the risk of chronic diseases such as diabetes, whereas consuming flour with a high content of resistant starch has a positive effect on regulating blood glucose levels. Given the generally positive correlation between resistant starch and amylose, increasing the amylose content in wheat germplasm has become a goal for quality improvement breeding research. 【Method】Four gene fragments of starch branching enzyme (TaSBEIIb) were selected to successfully construct a high-efficiency dsRNA expression vector. A gradient optimization based on culture medium components significantly enhanced dsRNA yield. Based on this, the effects of naked dsRNA and dsRNA encapsulated with the nanocarrier hydroxypropyltrimethyl ammonium chitosan chloride (HACC) on wheat starch metabolism were explored through foliar spraying. Utilizing a wheat seedling culture system, the impact of dsRNA spraying on the amylose content in wheat seedlings and the expression of starch-related genes was observed. Furthermore, a field trial analyzed the effects of dsRNA spraying on the amylose content in mature wheat grains. The protective effect of chitosan quaternary ammonium salt-coated dsRNA and its influence on amylose content in mature wheat grains were also investigated. 【Result】Four recombinant plasmids (pRNAI-TaSBE1-pRNAI-TaSBE4), expressing dsRNA were successfully constructed. The optimized fermentation medium increased the dsRNA yield from 26.54 mg·L^-1 to 50.65 mg·L^-1, representing a 91% increase compared to the initial medium. Spraying dsRNA interfered with the expression of the target genes, with the highest interference efficiency observed on day 7 for the TaSBEIIb1 fragment. After interference with the four fragments, the expression of TaSBEIIb was reduced by an average of 47.73%. Additionally, the interference of TaSBEIIb affected the expression of other genes in the starch synthesis pathway, including TaSSII, TaSSIV, and TaSBEIIa1 with peak interference efficiencies occurring on days 3, 7, and 3, respectively. Their expression levels decreased by an average of 54.53%, 59.94%, and 47.64%. The 2023 field trial indicated that spraying naked dsRNA increased the amylose content in wheat grains by 17.2%-36.5% after 7 days of treatment, although the effect diminished to 0.2%-8.3% by the maturity stage. In the 2024 field trial, multiple applications of both naked dsRNA and chitosan quaternary ammonium salt-coated raised the amylose content in mature wheat grains from 27.72% to 30.37%, about 10% increase compared to the control. 【Conclusion】Exogenous spraying of TaSBEIIbs-dsRNA effectively increases the amylose content in starch.

Key words: wheat, dsRNA, starch branching enzyme, amylose, crop quality improvement

李林燕, 张高阳, 冯先阳, 顾世龙, 黄烨楠, 孙忠科, 李成伟. 叶面喷施TaSBEIIbs-dsRNA提高小麦直链淀粉含量[J]. 中国农业科学, 2025, 58(22): 4557-4569.

LI LinYan, ZHANG GaoYang, FENG XianYang, GU ShiLong, HUANG YeNan, SUN ZhongKe, LI ChengWei. Foliar Spraying TaSBEIIbs-dsRNA to Increase Amylose Content in Wheat[J]. Scientia Agricultura Sinica, 2025, 58(22): 4557-4569.

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引物名称 Names of primers	引物序列 Primer of sequence (5′-3′)	片段大小 Fragment size (bp)	用途 Use
TaSBE1-F	ctagtctagaGACCTCCATGATGTATACCCACCA	435	基因扩增 Gene amplification
TaSBE1-R	acgcgtcgacAGAGCCATGAAATCATACATATCCTT	435
TaSBE2-F	ctagtctagaATAATGGCAATCCAAGAG	427
TaSBE2-R	acgcgtcgacCATGGTAGCTCCCTGTAA	427
TaSBE3-F	ctagtctagaGCGGTTACGAGAAGTTTG	459
TaSBE3-R	acgcgtcgacGCACCTCATCCCGAAAGT	459
TaSBE4-F	ctagtctagaTCGGAGGTTCTGGATGGC	422
TaSBE4-R	acgcgtcgaCATCCATGCCTCCTTTGTG	422
qSBEIIb1-F	CCAAGAGGCCCACAAGTA	104	qPCR
qSBEIIb1-R	AGAAATTCTGCATCACCC	104	qPCR
qSSII-F	TTTATGGTGGAGACCGAACAG	114	qPCR
qSSII-R	CAAGATTTCCATCACCGTAGC	114	qPCR
qSSIV-F	GGAAGCCCGAGGTCTGGAAA	81	qPCR
qSSIV-R	CGTACTGCGAAGCCGAGGTG	81	qPCR
qSBEIIa1-F	ACTGCTGCCGCGATCCGG	134	qPCR
qSBEIIa1-R	CACCGTCAGGCACCAGGACC	134	qPCR
GAPDH-F	CTGTTAGACTTGCGAAGCCA	103	qPCR
GAPDH-R	TCCTCATCAACGTAACCCAA	103	qPCR