Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (22): 4557-4569.doi: 10.3864/j.issn.0578-1752.2025.22.001

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Foliar Spraying TaSBEIIbs-dsRNA to Increase Amylose Content in Wheat

LI LinYan(), ZHANG GaoYang(), FENG XianYang, GU ShiLong, HUANG YeNan, SUN ZhongKe, LI ChengWei()   

  1. School of Biological Engineering, Henan University of Technology, Zhengzhou 450001
  • Received:2025-04-24 Accepted:2025-06-19 Online:2025-11-16 Published:2025-11-21
  • Contact: ZHANG GaoYang, LI ChengWei

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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

Table 1

Primers used in the experiment"

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
TaSBE2-F ctagtctagaATAATGGCAATCCAAGAG 427
TaSBE2-R acgcgtcgacCATGGTAGCTCCCTGTAA
TaSBE3-F ctagtctagaGCGGTTACGAGAAGTTTG 459
TaSBE3-R acgcgtcgacGCACCTCATCCCGAAAGT
TaSBE4-F ctagtctagaTCGGAGGTTCTGGATGGC 422
TaSBE4-R acgcgtcgaCATCCATGCCTCCTTTGTG
qSBEIIb1-F CCAAGAGGCCCACAAGTA 104 qPCR
qSBEIIb1-R AGAAATTCTGCATCACCC
qSSII-F TTTATGGTGGAGACCGAACAG 114 qPCR
qSSII-R CAAGATTTCCATCACCGTAGC
qSSIV-F GGAAGCCCGAGGTCTGGAAA 81 qPCR
qSSIV-R CGTACTGCGAAGCCGAGGTG
qSBEIIa1-F ACTGCTGCCGCGATCCGG 134 qPCR
qSBEIIa1-R CACCGTCAGGCACCAGGACC
GAPDH-F CTGTTAGACTTGCGAAGCCA 103 qPCR
GAPDH-R TCCTCATCAACGTAACCCAA

Fig. 1

Cloning of four gene fragments of TaSBEIIb and construction of recombinant plasmid A: Amplification of target gene; B: Verification of recombinant plasmid by double enzyme digestion; C: DSRNA extraction and double enzyme (DNase I and RNase A) digestion verification; D: Map of pRNAi-TaSBE1-pRNAi-TaSBE4 dsRNA expression vector. M: DNA marker; 1: SBE1; 2: SBE2; 3: SBE3; 4: SBE4; 5: pRNAi-SBE1 double enzyme digestion; 6: pRNAi-SBE2 double enzyme digestion; 7: pRNAi-SBE3 double enzyme digestion; 8: pRNAi-SBE4 double enzyme digestion; 9: Obtaining dsTaSBE1 by thermal cracking; 10: Obtaining dsTaSBE2 by thermal cracking; 11: Obtaining dsTaSBE3 by thermal cracking; 12: Obtaining dsTaSBE4 by thermal cracking; 13: dsTaSBE1 after double enzyme digestion; 14: dsTaSBE2 after double enzyme digestion; 15: dsTaSBE3 after double enzyme digestion; 16: dsTaSBE4 after double enzyme digestion"

Fig. 2

Optimization result diagram of fermentation medium A: The yield of dsRNA in different media; B: The growth curve of 48 h fermentation with different concentrations of yeast powder; C: The yield of dry cell weight and dsRNA after 48 h fermentation with different concentrations of yeast powder; D: The growth curve of peptone fermentation with different concentrations for 48 h; E: The yield of dry cell weight and dsRNA after peptone fermentation with different concentrations for 48 h; F: Growth curve of phosphate fermentation with different concentrations for 48 h; G: The output of dry cell weight and dsRNA after 48 h of phosphate fermentation with different concentrations. The number before "/"indicates the concentration of K2HPO4, and the number after “/” indicates the concentration of KH2PO4; H: The growth curve of glycerol fermentation with different concentrations for 48 h; I: The yield of dry cell weight and dsRNA after 48 h fermentation with different concentrations of glycerol; J: Results before and after optimization of culture medium. Different lowercase letters indicate significant differences at P<0.05 level. The same as below"

Fig. 3

Amylose content and expression level of starch synthesis related genes in wheat leaves A: Establishing amylose standard curve by perchloric acid method; B: Amylose content in wheat leaves; C: TaSBEIIb1; D: TaSSII; E: TaSSIV; F: TaSBEIIa1"

Fig. 4

Amylose content in wheat grain A: Amylose standard curve established by iodine binding method; B: Amylose content in wheat grain; C: The content of amylose in wheat grain after spraying naked dsRNA; D: The content of amylose in wheat grain after spraying HACC-dsRNA nanocomposite"

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