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.

图/表 5

表1

图1

图2

图3

图4

参考文献 45

[1]	孙月, 杨慧玉, 朱文根, 王小燕, 唐益苗. 小麦Whirly基因家族的鉴定及表达分析. 农业生物技术学报, 2023, 31(2): 223-231.
	SUN Y, YANG H Y, ZHU W G, WANG X Y, TANG Y M. Identification and expression analysis of wheat (Triticum aestivum) whirly gene family. Journal of Agricultural Biotechnology, 2023, 31(2): 223-231. (in Chinese)
[2]	李雪, 吕新业. 现阶段中国粮食安全形势的判断: 数量和质量并重. 农业经济问题, 2021, 42(11): 31-44.
	LI X, LÜ X Y. Judgment of China’s food security situation at the present stage: Pay equal attention to quantity and quality. Issues in Agricultural Economy, 2021, 42(11): 31-44. (in Chinese)
[3]	秦宇, 马硕晗, 徐恩波, 周建伟, 陈健初, 刘东红, 叶兴乾. 绿色加工对全谷物结构及膳食纤维、酚类物质影响的研究进展. 食品与生物技术学报, 2022, 41(11): 10-21.
	QIN Y, MA S H, XU E B, ZHOU J W, CHEN J C, LIU D H, YE X Q. Structural reorganization of whole grains with dietary fiber and phenols change during green processes. Journal of Food Science and Biotechnology, 2022, 41(11): 10-21. (in Chinese)
[4]	ZHANG D P, KISHIMOTO N. Quantum chemical investigation into the structural analysis and calculated Raman spectra of amylose modeled with linked glucose molecules. Molecules, 2024, 29(12): 2842. doi: 10.3390/molecules29122842
[5]	LI M, DHITAL S, WEI Y M. Multilevel structure of wheat starch and its relationship to noodle eating qualities. Comprehensive Reviews in Food Science and Food Safety, 2017, 16(5): 1042-1055. doi: 10.1111/crf3.2017.16.issue-5
[6]	SUN Z Z, SUN X X, GE X Z, LU Y F, ZHANG X Y, SHEN H S, YU X Z, ZENG J, GAO H Y, LI W H. Structural, rheological, pasting, and digestive properties of wheat A-starch: Effect of outshell removal combined with annealing. International Journal of Biological Macromolecules, 2023, 244: 125401. doi: 10.1016/j.ijbiomac.2023.125401
[7]	CORRADO M, ZAFEIRIOU P, AHN-JARVIS J H, SAVVA G M, EDWARDS C H, HAZARD B A. Impact of storage on starch digestibility and texture of a high-amylose wheat bread. Food Hydrocolloids, 2023, 135: 108139. doi: 10.1016/j.foodhyd.2022.108139
[8]	董喆, 赵阳, 孙姗姗, 曹进, 罗娇依, 李梦怡. 肠道微生物组对抗性淀粉的降解作用研究进展. 食品安全质量检测学报, 2024, 15(24): 96-102.
	DONG Z, ZHAO Y, SUN S S, CAO J, LUO J Y, LI M Y. Advancements in research on the degradation of resistant starch by gut microbiome. Journal of Food Safety & Quality, 2024, 15(24): 96-102. (in Chinese)
[9]	FENG T T, WANG L L, LI L Y, LIU Y, CHONG K, THEIßEN G, MENG Z. OsMADS14 and NF-YB1 cooperate in the direct activation of OsAGPL2 and Waxy during starch synthesis in rice endosperm. New Phytologist, 2022, 234(1): 77-92. doi: 10.1111/nph.17990 pmid: 35067957
[10]	LAI Y C, WANG S Y, GAO H Y, NGUYEN K M, NGUYEN C H, SHIH M C, LIN K H. Physicochemical properties of starches and expression and activity of starch biosynthesis-related genes in sweet potatoes. Food Chemistry, 2016, 199: 556-564. doi: 10.1016/j.foodchem.2015.12.053 pmid: 26776008
[11]	SORNDECH W, SAGNELLI D, MEIER S, JANSSON A M, LEE B H, HAMAKER B R, ROLLAND-SABATÉ A, HEBELSTRUP K H, TONGTA S, BLENNOW A. Structure of branching enzyme- and amylomaltase modified starch produced from well-defined amylose to amylopectin substrates. Carbohydrate Polymers, 2016, 152: 51-61. doi: S0144-8617(16)30772-X pmid: 27516249
[12]	JIANG L L, YU X M, QI X, YU Q, DENG S, BAI B, LI N, ZHANG A, ZHU C F, LIU B, PANG J S. Multigene engineering of starch biosynthesis in maize endosperm increases the total starch content and the proportion of amylose. Transgenic Research, 2013, 22(6): 1133-1142. doi: 10.1007/s11248-013-9717-4 pmid: 23740205
[13]	GURUNATHAN S, RAMADOSS B R, MUDILI V, SIDDAIAH C, KALAGATUR N K, BAPU J R K, MOHAN C D, ALQARAWI A A, HASHEM A, ABD ALLAH E F. Single nucleotide polymorphisms in starch biosynthetic genes associated with increased resistant starch concentration in rice mutant. Frontiers in Genetics, 2019, 10: 946. doi: 10.3389/fgene.2019.00946 pmid: 31803220
[14]	LI H T, YU W W, DHITAL S, GIDLEY M J, GILBERT R G. Starch branching enzymes contributing to amylose and amylopectin fine structure in wheat. Carbohydrate Polymers, 2019, 224: 115185. doi: 10.1016/j.carbpol.2019.115185
[15]	GAO M, FISHER D K, KIM K N, SHANNON J C, GUILTINAN M J. Independent genetic control of maize starch-branching enzymes IIa and IIb. Isolation and characterization of a Sbe2a cDNA. Plant Physiology, 1997, 114(1): 69-78. pmid: 9159942
[16]	LI J Y, JIAO G A, SUN Y W, CHEN J, ZHONG Y X, YAN L, JIANG D, MA Y Z, XIA L Q. Modification of starch composition, structure and properties through editing of TaSBEIIa in both winter and spring wheat varieties by CRISPR/Cas9. Plant Biotechnology Journal, 2021, 19(5): 937-951. doi: 10.1111/pbi.13519 pmid: 33236499
[17]	TOUZDJIAN P, KOHLRAUSCH TÁVORA F, DE ASSIS DOS SANTOS DINIZ F, DE MORAES RÊGO-MACHADO C, CHAGAS FREITAS N, BARBOSA MONTEIRO ARRAES F, CHUMBINHO DE ANDRADE E, FURTADO L L, OSIRO K O, LIMA DE SOUSA N, et al. CRISPR/cas- and topical RNAi-based technologies for crop management and improvement: Reviewing the risk assessment and challenges towards a more sustainable agriculture. Frontiers in Bioengineering and Biotechnology, 2022, 10: 913728. doi: 10.3389/fbioe.2022.913728
[18]	ZHAO J, LIU N, MA J, HUANG L N, LIU X N. Effect of silencing CYP6B6 of Helicoverpa armigera (Lepidoptera: Noctuidae) on its growth, development, and insecticide tolerance. Journal of Economic Entomology, 2016, 109(6): 2506-2516. doi: 10.1093/jee/tow181
[19]	GREGORIOU M E, MATHIOPOULOS K D. Knocking down the sex peptide receptor by dsRNA feeding results in reduced oviposition rate in olive fruit flies. Archives of Insect Biochemistry and Physiology, 2020, 104(2): e21665. doi: 10.1002/arch.v104.2
[20]	TAKAHASHI T, UI-TEI K. Mutual regulation of RNA silencing and the IFN response as an antiviral defense system in mammalian cells. International Journal of Molecular Sciences, 2020, 21(4): 1348. doi: 10.3390/ijms21041348
[21]	WANG C Y, GUO K. Application of dsRNA in the pine wood nematode, Bursaphelenchus xylophilus. Methods in Molecular Biology, 2024, 2771: 133-139. doi: 10.1007/978-1-0716-3702-9_18 pmid: 38285400
[22]	WERNER B T, GAFFAR F Y, SCHUEMANN J, BIEDENKOPF D, KOCH A M. RNA-spray-mediated silencing of Fusarium graminearum AGO and DCL genes improve barley disease resistance. Frontiers in Plant Science, 2020, 11: 476. doi: 10.3389/fpls.2020.00476
[23]	PALLIS S, ALYOKHIN A, MANLEY B, RODRIGUES T, BARNES E, NARVA K. Effects of low doses of a novel dsRNA-based biopesticide (calantha) on the Colorado potato BeetleFree. Journal of Economic Entomology, 2023, 116(2): 456-461. doi: 10.1093/jee/toad034 pmid: 36895198
[24]	XU X, JIAO Y B, SHEN L L, LI Y, MEI Y P, YANG W G, LI C Q, CAO Y, CHEN F L, LI B, YANG J G. Nanoparticle-dsRNA treatment of pollen and root systems of diseased plants effectively reduces the rate of tobacco mosaic virus in contemporary seeds. Applied Materials & Interfaces, 2023, 15(24): 29052-29063.
[25]	张浩然, 刘晓莹, 周峰龙, 许明晨, 郭争争, 程佳雨, 张坤普, 王道文, 师翠兰. TaGW2-A1过表达小麦种质的创制及表型分析. 河南农业科学, 2025, 54(1): 1-8.
	ZHANG H R, LIU X Y, ZHOU F L, XU M C, GUO Z Z, CHENG J Y, ZHANG K P, WANG D W, SHI C L. Creation and phenotypic analysis of TaGW2-A1-overexpression transgenic wheat germplasm. Journal of Henan Agricultural Sciences, 2025, 54(1): 1-8. (in Chinese)
[26]	FENG X Y, SHI Y N, SUN Z K, LI L Y, IMRAN M, ZHANG G Y, ZHANG G Y, LI C W. Control of Fusarium graminearum infection in wheat by dsRNA-based spray-induced gene silencing. Journal of Agricultural and Food Chemistry, 2025, 73(20): 12146-12155. doi: 10.1021/acs.jafc.4c12665
[27]	FAJARDO D, JAYANTY S S, JANSKY S H. Rapid high throughput amylose determination in freeze dried potato Tuber samples. Journal of Visualized Experiments, 2013(80): 50407.
[28]	PARK S H, BEAN S R, WILSON J D, SCHOBER T J. Rapid isolation of sorghum and other cereal starches using sonication. Cereal Chemistry, 2006, 83(6): 611-616. doi: 10.1094/CC-83-0611
[29]	KAUFMAN R C, WILSON J D, BEAN S R, HERALD T J, SHI Y C. Development of a 96-well plate iodine binding assay for amylose content determination. Carbohydrate Polymers, 2015, 115: 444-447. doi: 10.1016/j.carbpol.2014.09.015 pmid: 25439917
[30]	JIANG X, ATTIOGBE K B, GUO Y T, WU X Y. Production of double-stranded RNA using the prokaryotic promoter-mediated bidirectional transcription. Methods in Molecular Biology, 2024, 2771: 47-55. doi: 10.1007/978-1-0716-3702-9_8 pmid: 38285390
[31]	崔锦程, 崔洁, 卞小莹. 生产双链RNA工程菌的研究进展. 生物工程学报, 2025, 41(2): 546-558.
	CUI J C, CUI J, BIAN X Y. Research progress in the engineering strains for producing double-stranded RNA. Chinese Journal of Biotechnology, 2025, 41(2): 546-558. (in Chinese)
[32]	余金兰, 何倩, 李欣茹, 谭红晓, 路来风. 原核表达dsRNA-StPPO发酵工艺优化及应用. 食品与发酵工业, 2025, 51(2): 59-67. doi: 10.13995/j.cnki.11-1802/ts.038465
	YU J L, HE Q, LI X R, TAN H X, LU L F. Optimization and application of prokaryotic expression of dsRNA-StPPO fermentation process. Food and Fermentation Industries, 2025, 51(2): 59-67. (in Chinese)
[33]	常瑞, 王俊, 付开赟, 廖兰兰, 丁新华, 何江, 郭文超, 吐尔逊·阿合买提, 任羽. 4种原核表达双链RNA的dsRNA提取方法效果评价. 新疆农业科学, 2021, 58(4): 700-711. doi: 10.6048/j.issn.1001-4330.2021.04.013
	CHANG R, WANG J, FU K Y, LIAO L L, DING X H, HE J, GUO W C, TOULXUN A, REN Y. Comparative study on the effect of 4 kind of dsRNA extraction methods form prokaryotic expression double-stranded RNA. Xinjiang Agricultural Sciences, 2021, 58(4): 700-711. (in Chinese) doi: 10.6048/j.issn.1001-4330.2021.04.013
[34]	DA ROSA J, VIANA A J C, FERREIRA F R A, KOLTUN A, MERTZ-HENNING L M, MARIN S R R, RECH E L, NEPOMUCENO A L. Optimizing dsRNA engineering strategies and production in E. coli HT115 (DE3). Journal of Industrial Microbiology & Biotechnology, 2024, 51: kuae028.
[35]	姜晨, 吴昕曈, 陈慧玲, 刘乐, 张谦, 李宪臻, 郭小宇. 重组蔗糖异构酶在枯草芽孢杆菌中的表达及发酵优化. 生物学杂志, 2024, 41(6): 39-46.
	JIANG C, WU X T, CHEN H L, LIU L, ZHANG Q, LI X Z, GUO X Y. Expression and fermentation optimization of recombinant sucrose isomerase in Bacillus subtilis. Journal of Biology, 2024, 41(6): 39-46. (in Chinese)
[36]	曹倩荣, 刘春卯, 郑翔, 贾振华, 吴芳彤. 蛋白酶枯草芽孢杆菌工程菌株WB800N/pHT43-npr-PrsA的发酵工艺优化. 饲料研究, 2024, 47(14): 100-106.
	CAO Q R, LIU C M, ZHENG X, JIA Z H, WU F T. Optimization of fermentation process for Bacillus subtilis WB800N/pHT43-npr-PrsA recombinant strain with protease activity. Feed Research, 2024, 47(14): 100-106. (in Chinese)
[37]	符长春, 杨瑞金, 仝艳军. Bifidobacterium bifidum ATCC 29521 β-半乳糖苷酶的生物信息学分析、异源表达及其酶学性质. 食品科学, 2025, 46(6): 98-107.
	FU C C, YANG R J, TONG Y J. Bioinformatics analysis, heterologous expression and enzymatic properties of β-galactosidase from Bifidobacterium bifidum ATCC 29521. Food Science, 2025, 46(6): 98-107. (in Chinese)
[38]	SUN Y W, JIAO G A, LIU Z P, ZHANG X, LI J Y, GUO X P, DU W M, DU J L, FRANCIS F, ZHAO Y D, XIA L Q. Generation of high- amylose rice through CRISPR/Cas9-mediated targeted mutagenesis of starch branching enzymes. Frontiers in Plant Science, 2017, 8: 298.
[39]	徐婉莹, 戚晓雪, 徐莹, 汪东风. 基于培养基优化提高过表达MET14基因的重组库德毕赤酵母M48脱镉能力的研究. 食品工业科技, 2022, 43(19): 288-297.
	XU W Y, QI X X, XU Y, WANG D F. Study on improving the cadmium removal ability of recombinant Pichia kudriavzevii M48 overexpressed MET14 gene based on medium optimization. Science and Technology of Food Industry, 2022, 43(19): 288-297. (in Chinese)
[40]	SIMON I, PERSKY Z, AVITAL A, HARAT H, SCHROEDER A, SHOSEYOV O. Foliar application of dsRNA targeting endogenous potato (Solanum tuberosum) isoamylase genes ISA1 ISA2 and ISA3 confers transgenic phenotype. International Journal of Molecular Sciences, 2023, 24(1): 190. doi: 10.3390/ijms24010190
[41]	REGINA A, BIRD A, TOPPING D, BOWDEN S, FREEMAN J, BARSBY T, KOSAR-HASHEMI B, LI Z Y, RAHMAN S, MORELL M. High-amylose wheat generated by RNA interference improves indices of large-bowel health in rats. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(10): 3546-3551.
[42]	FERREIRA S J, SENNING M, FISCHER-STETTLER M, STREB S, AST M, NEUHAUS H E, ZEEMAN S C, SONNEWALD S, SONNEWALD U. Simultaneous silencing of isoamylases ISA1, ISA2 and ISA3 by multi-target RNAi in potato tubers leads to decreased starch content and an early sprouting phenotype. PLoS ONE, 2017, 12(7): e0181444.
[43]	QIAO H, ZHAO J, WANG X F, XIAO L B, ZHU-SALZMAN K, LEI J X, XU D J, XU G C, TAN Y A, HAO D J. An oral dsRNA delivery system based on chitosan induces G protein-coupled receptor kinase 2 gene silencing for Apolygus lucorum control. Pesticide Biochemistry and Physiology, 2023, 194: 105481. doi: 10.1016/j.pestbp.2023.105481
[44]	XU X, YU T T, ZHANG D S, SONG H P, HUANG K, WANG Y, SHEN L L, LI Y, WANG F L, ZHANG S B, JIAO Y B, YANG J G. Evaluation of the anti-viral efficacy of three different dsRNA nanoparticles against potato virus Y using various delivery methods. Ecotoxicology and Environmental Safety, 2023, 255: 114775. doi: 10.1016/j.ecoenv.2023.114775
[45]	李林燕, 张高阳, 刘星雨, 孙千惠, 孙忠科, 李成伟, 罗德平. 外源dsRNA在植物抗病虫害中的应用研究进展. 农药, 2025, 64(07): 469-479.
	LI L Y, ZHANG G Y, LIU X Y, SUN Q H, SUN Z K, LI C W, LUO D P. Research progress on the application of exogenous dsRNA in plant disease and pest resistance. Agrochemicals, 2025: 64(07): 469-479. (in Chinese)

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