籼稻明恢86多基因遗传转化体系的研究

魏林艳; 连玲; 魏毅东; 罗曦; 何炜; 谢鸿光; 谢华安; 张建福

doi:10.19303/j.issn.1008-0384.2019.06.001

籼稻明恢86多基因遗传转化体系的研究

doi: 10.19303/j.issn.1008-0384.2019.06.001

魏林艳^{1, 2, 3,},
连玲^{1, 2, 3},
魏毅东^{1, 2, 3},
罗曦^{1, 2, 3},
何炜^{1, 2, 3},
谢鸿光^{1, 2, 3},
谢华安^{1, 2, 3, ,},
张建福^{1, 2, 3, ,}

1.
福建省农业科学院水稻研究所, 福建福州 350019
2.
农业部华南杂交水稻种质创新与分子育种重点实验室/福州(国家)水稻改良分中心/福建省作物分子育种工程实验室/福建省作物种质创新与分子育种省部共建国家重点实验室培育基地/杂交水稻国家重点实验室华南研究基地, 福建福州 350003
3.
水稻国家工程实验室, 福建福州 350003

基金项目:

福建省科技计划项目——省属公益类科研院所基本科研专项 2016R1020-12

国家重点研发计划项目 2017YFD0100100

详细信息

作者简介:
魏林艳(1988-), 女, 硕士, 研究实习员, 主要从事水稻遗传转化研究(E-mail:290689712@qq.com)

通讯作者:
谢华安(1941-), 男, 研究员, 主要从事杂交水稻育种研究(E-mail:huaanxie@163.com)

张建福(1971-), 男, 博士, 研究员, 主要从事水稻分子设计育种研究(E-mail:jianfzhang@163.com)

中图分类号: S511
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- 被引次数: 0
出版历程
- 收稿日期: 2019-02-19
- 修回日期: 2019-05-05
- 刊出日期: 2019-06-28

Multi-gene Transformation of Indica Rice, Minghui 86

WEI Lin-yan^{1, 2, 3
,},
LIAN Ling^{1, 2, 3},
WEI Yi-dong^{1, 2, 3},
LUO Xi^{1, 2, 3},
HE Wei^{1, 2, 3},
XIE Hong-guang^{1, 2, 3},
XIE Hua-an^{1, 2, 3
, ,},
ZHANG Jian-fu^{1, 2, 3
, ,}

1.
Rice Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350019, China
2.
Key Laboratory of Germplasm Innovation and Molecular Breeding of Hybrid Rice for South China, Ministry of Agriculture and Rual/Fuzhou Branch, National Rice Improvement Center of China/Fujian Engineering Laboratory of Crop Molecular Breeding/Incubator of National Key Laboratory of Crops Germplasm Innovation and Molecular Breeding Between Fujian and Ministry of Science and Technology/Base for South-China, National Key Laboratory of Hybrid Rice for China, Fuzhou, Fujian 350003, China
3.
National Engineering Laboratory of Rice of China, Fuzhou, Fujian 350003, China

摘要

摘要: 目的探索适合籼稻明恢86多基因遗传转化的条件，为创制含高产、抗逆、抗虫、抗病等基因的水稻新材料奠定基础。方法以籼稻明恢86为受体材料，将构建好的含作物高产基因RRM2、耐旱基因HS1、抗除草剂基因EPSPS、抗虫基因Bt、细胞凋亡抑制基因iap和促细胞再生基因p35等多基因载体（载体分别命名为P5和P8）进行遗传转化。在此基础上，分别对多基因遗传转化体系的受体材料、农杆菌浓度、侵染时间、共培养方式、G418筛选浓度和草甘膦筛选浓度等主要影响因素进行试验，探讨其适宜的转化条件。结果受体材料的筛选结果表明，幼胚的出愈率显著高于成熟胚，且其愈伤组织状态相对较好；各转化条件的筛选结果，农杆菌侵染浓度OD₆₀₀为0.4~0.6、侵染时间15~20 min、共培养2~3 d、培养基上添加无菌滤纸、G418筛选浓度150 mg·L^-1和草甘膦筛选浓度800 mg·L^-1是提高转化效率的优化条件；PCR分析结果，多基因载体P5中的GUS基因成功转入籼稻明恢86。结论通过对培养条件的优化，可使籼稻明恢86愈伤组织的诱导愈伤率和抗性愈伤率得到显著提高。
- 基因 /
- 遗传转化 /
- 籼稻 /
- 明恢86
Abstract: Objective Conditions for simultaneous transformation of multiple genes in Indica rice, Minghui 86, were studied to facilitate the development of high yield and resistant breeds. Method Two multi-gene vectors designated as P5 and P8 that harbored the high-yield RRM2, drought-tolerant HS1, herbicide-resistant EPSPS, and insect-resistant Bt as well as the apoptosis-inhibiting iap and gene that promotes cell regeneration p35 were transformed to the receptor Minghui 86 using agrobacterium-based transformation methodology. The receptor, callus culture, agrobacterium concentration, infection time, co-culture, and screening concentrations of G418 and glyphosate were evaluated for the selection. Result Under same culture conditions, the recovery rate of Minghui 86 immature embryos was significantly higher with a better callus quality than that of the mature embryos. The optimal transformation conditions were determinated to be a bacterial concentration of OD₆₀₀=0.4-0.6, an infection time of 15-20 m, co-culture for 2-3 d with the addition of sterile filter paper in medium, and the screening concentration of G418 at 150 mg·L^-1 and of glyphosate at 800 mg·L^-1. The PCR detection confirmed that GUS in the polygenic vector P5 was successfully transferred into Indica cv. Minghui 86. Conclusion The optimized culture conditions afforded significantly improved callus and resistance induction rates in Minghui 86.
- gene /
- genetic transformation /
- indica rice /
- Minghui 86

HTML全文

图 1 多基因表达载体结构

注：A代表P5载体结构图；B代表P8载体结构图。

Figure 1. Construct of multi-gene expression vector

Note:A=The construct of multi-gene P5;B=The construct of multi-gene P8.

下载: 全尺寸图片幻灯片

图 2 明恢86幼胚和成熟胚愈伤组织诱导情况

注：A为明恢86幼胚，B为明恢86成熟胚。

Figure 2. Callus induction of immature and mature embryos of Minghui 8

Note:A.Minghui 86immature embryos, B.Minghui 86mature embryo.

下载: 全尺寸图片幻灯片

图 3 草甘膦对明恢86愈伤组织筛选浓度的确定

注：A~D分别为草甘膦400、600、800、1 000 mg·L^-1。

Figure 3. Determination of screening concentration of glyphosate on callus of Minghui 86

Note:A-D=400, 600, 800, 1 000 mg·L^-1.

下载: 全尺寸图片幻灯片

图 4 G418对明恢86愈伤组织筛选浓度的确定

注：A~D分别为G418=0、100、150、200 mg·L^-1G418。

Figure 4. Determination of screening concentration of G418 on callus of Minghui 86

Note:A-D:418=0, 100, 150, 200 mg·L^-1.

下载: 全尺寸图片幻灯片

图 5 明恢86的多基因遗传转化

注：A为继代；B为筛选；C为预分化；D为分化；E为生根；F为炼苗。

Figure 5. Multi-gene transformation of Minghui 86

Note:A=Subculture; B=Screening; C=Predifferentiation; D=Differentiation; E=Rooting; F=Refining.

下载: 全尺寸图片幻灯片

图 6 转基因植株的PCR检测

注：M为2 000 bp；1为水；2为非转基因植株；3~9为转基因植株；10为质粒。

Figure 6. PCR analysis on transgenic plan

Note:M=2 000 bp; 1=H₂O; 2=Non-transgenic plants; 3-9=Transgenic plants; 10=Plasmid.

下载: 全尺寸图片幻灯片

表 1 农杆菌浓度对转化效率的影响

Table 1. Transformation rate affected by agrobacterium screening concentration

农杆菌浓度(OD₆₀₀值) Agrobacterium concentration	抗性愈伤组织数 No. of resistance callus (Mean±SD)	抗性愈伤组织率 Resistance callus induction rate/% (Mean±SD)	愈伤组织生长状态 Callus grow status
0.20	9.33±1.15dD	23.33±2.89dD	无农杆菌No visible bacteria colony
0.40	24.00±2.00bB	60.00±5.00bB	无农杆菌No visible bacteria colony
0.60	30.00±2.00aA	75.00±5.00aA	少量农杆菌Few visible bacteria colonies
0.80	17.33±1.15cC	43.33±2.89cC	大量农杆菌Many visible bacteria colonies
注：表中同列数值后无相同小、大写字母者分别表示处理间差异达显著(P < 0.05)或极显著(P < 0.01)水平。表 2~4同。 Note：Different lowercase letters after the value indicate significant difference (P < 0.01).The same as Table 2-4.

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表 2 不同侵染时间对抗性愈伤率的影响

Table 2. Effect of infecting time on antagonistic callus rate

侵染时间 Infectiontime /min	抗性愈伤数 No. of resistance callus (Mean±SD)	抗性愈伤率 Resistance callus induction rate/% (Mean±SD)	愈伤生长状态 Callus grow status
5	3.33±0.58dD	16.67±2.89dD	无褐化No browning
10	6.67±1.53cdCD	33.33±7.64cdCD	少量褐化Slighty browning
15	11.67±1.53aA	58.33±7.64aA	边缘，顶部褐化Browning at the margin and top
20	12.33±1.53aA	61.67±7.64aA	边缘，顶部褐化Browning at the margin and top
25	10.33±1.15abAB	51.67±5.77abAB	褐化严重Severely Browning
30	7.33±0.58bcBC	36.67±2.89bcBC	褐化严重Severely Browning

下载: 导出CSV

表 3 共培养方式的转化效果

Table 3. Transformation effect of co-culture

共培养方式 Co-cultivation method	愈伤数 No. of callus	抗性愈伤数 No. of resistance callus (Mean±SD)	抗性愈伤率 Resistance callus induction rate/% (Mean±SD)	愈伤生长状态 Callus grow status
不加滤纸 Without filter paper	40	17.66±2.52bB	44.33±6.03bB	生长慢，易褐化 Growing slowly and prone to browning
加滤纸 With filter paper	40	30.33±2.52aA	76.00±6.56aA	生长快，褐化慢 Growing fast and resistant to Browning

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表 4 共培养时间对抗性愈伤率的影响

Table 4. Effect of co-culture time on callus resistance

共培养时间 Co-culture time/d	侵染愈伤数 Number of contaminated calli (Mean)	抗性愈伤数 Number of resistant calli (Mean)	抗性愈伤率 Frequency of resistant calli/% (Mean±SD)	愈伤生长状态 Callus grow status
1	56.67	20.33	35.96±1.10dD	无农杆菌 No visible bacteria colony
2	70.33	54.33	77.26±1.07bB	底部偶见少量农杆菌 Occasionally bacteria colony at the bottom
3	72.67	60.00	82.67±1.15aA	底部有明显农杆菌 Visible bacteria colony at the bottom
4	58.33	39.33	67.33±1..53cC	大量农杆菌包裹 Covered with bacteria

下载: 导出CSV

参考文献(25)

[1]	王维旭, 张骥诚, 刘学群, 等.籼稻品种Kasalath遗传转化条件的研究[J].安徽农业科学, 2010, 38(4):1735-1737, 1856. doi: 10.3969/j.issn.0517-6611.2010.04.031 WANG W X, ZHANG J C, LIU X Q, et al.Study on the Factors of Genetic Transformation for Indica Rice Kasalath[J]. Journal of Anhui Agri.Sci.2010, 38(4):1735-1737, 1856. (in Chinese) doi: 10.3969/j.issn.0517-6611.2010.04.031
[2]	柳红.籼稻高效遗传转化体系的建立[D].福州: 福建农林大学, 2009: 1-75. http://cdmd.cnki.com.cn/Article/CDMD-10389-2009170697.htm LIU H.Establishment of Indica efficient genetic transformationsystem[D].Fuzhou: Fujian Agriculture and Forestry University, 2009: 1-75. (in Chinese) http://cdmd.cnki.com.cn/Article/CDMD-10389-2009170697.htm
[3]	向阳, 赵德刚, 朱冬雪, 等.籼稻成熟胚愈伤组织诱导及不定芽分化[J].山地农业生物学报, 2004, 23(3):193-197. doi: 10.3969/j.issn.1008-0457.2004.03.002 XIANG Y, ZHAO D G, ZHU D X, et al.Indica rice matureembryo callus induction and adventitious bud differentiation[J].J Mountain Agric Biol, 2004, 23(3):193-197. (in Chinese) doi: 10.3969/j.issn.1008-0457.2004.03.002
[4]	BABA A, HASEZAWA S, SYONO K, et al. Cultivation of riceprotoplasts and their transformation mediated by Agrobacterium spheroplasts[J]. Plant Cell Physiol, 1986, 27:463-468.
[5]	HIEI Y, OHTA S, KOMARI T, et al. Efficient transformation of rice (Oryza sativa L.). mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA[J]. Plant J, 1994(6):271-282.
[6]	CHENG X, SARDANA R, KAPLAN H, et al.Agrobacterium-transformed rice plants expressing syntheticcryIA(a) and cry IA(c) genes are highly toxic to striped stem borer and yellow stem borer[J]. Proc Natl Acad Sci USA, 1998, 95:2767-2772. doi: 10.1073/pnas.95.6.2767
[7]	项友斌, 梁竹青, 高明尉, 等.农杆菌介导的苏云金杆菌抗虫因cryIA(b)和cryIA(c)在水稻中的遗传转化及蛋白表达[J].生物工程学报, 1999, 15(4):494-500. doi: 10.3321/j.issn:1000-3061.1999.04.015 XIANG Y B, LIANG Z Q, GAO M W, et al.Agrobacterium mediated Transformation of Insecticidal BacillusthuringiensiscryIA(b) and cryIA(c) Genes and Their Expression in Rice Chinese Journal of Biotechnology[J]. Journal of Bioengineering, 1999, 15(4):494-500. (in Chinese) doi: 10.3321/j.issn:1000-3061.1999.04.015
[8]	CHEN L, MARMEY P, TAYLOR N J, et al. Expression and inheritance of multiple transgenes in rice plants[J]. Nature Biotechnology, 1998, 16:1060-1064. doi: 10.1038/3511
[9]	冯道荣, 许新萍, 邱国华, 等.多个抗病抗虫基因在水稻中的遗传和表达[J].科学通报, 2000, 45(15):1593-1599. doi: 10.3321/j.issn:0023-074X.2000.15.004 FENG D R, XU X P, QIU G H, et al.Inheritance and expression of multiple disease resistance and insect resistance genes in rice[J].Chinese Science Bulletin, 2000, 45(15):1593-1599. (in Chinese) doi: 10.3321/j.issn:0023-074X.2000.15.004
[10]	YE X, AL-BABILI S, KLOTI A, et al. Engineering the provitamin A (β-Carotene)biosynthetic pathway into (Carotenoid-free) rice endosperm[J].Science, 2000, 287:303-305. doi: 10.1126/science.287.5451.303
[11]	李宝健, 朱华晨.论应用多基因转化策略综合改良生物体遗传性研究方向的前景[J].中山大学学报, 2005, 44(4):79-83. doi: 10.3321/j.issn:0529-6579.2005.04.021 LI B J, ZHU H C.Prospects for the application of multi-gene transformation strategies to comprehensively improve the genetic research direction of organisms[J].Journal of Sun Yat-sen University, 2005, 44(4):79-83. (in Chinese) doi: 10.3321/j.issn:0529-6579.2005.04.021
[12]	王慧中, 黄大年, 鲁瑞芳.转mt1D/gutD双价基因水稻的耐盐性[J].科学通报, 2000, 45(7):724-728. doi: 10.3321/j.issn:0023-074X.2000.07.012 WANG H Z, HUANG D N, LU R F.Salt tolerance of transgenic rice with mt1D/gutD[J].Chinese Science Bulletin, 2000, 45(7):724-728. (in Chinese) doi: 10.3321/j.issn:0023-074X.2000.07.012
[13]	朱永兴, 曹鹏, 许兴, 等.多基因表达载体KCTB转化宁夏枸杞的研究[J].中国科学通报, 2010, 26(9):74-77. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zgnxtb201009014 ZHU Y X, CAO P, XU X, et al.The Transformation of Multigene Expressed Vector for Lyciumbarbarum L.[J].Chinese Agricultural Science Bulletin, 2010, 26(9):74-77. (in Chinese) http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zgnxtb201009014
[14]	朱丽, 傅亚萍, 刘文真, 等.利用共转化和花药培养技术快速获得无选择标记的三价转基因水稻[J].中国水稻科学, 2007, 21(5):475-481. doi: 10.3321/j.issn:1001-7216.2007.05.006 ZHU L, FU Y P, LIU W Z, et al.Rapidly obtaining trivalent transgenic rice without selection marker by co-transformation and anther culture techniques[J].Chinese Rice Science, 2007, 21(5):475-481. (in Chinese) doi: 10.3321/j.issn:1001-7216.2007.05.006
[15]	CAO M X, HUANG J Q, WEI Z M, et al. Agrobacterium-mediated multiple-gene transformation in rice using a single vector[J]. J IntegrPlantBiol, 2005, 47(2):233-242.
[16]	NAQVI S, FARRE G, SANAHUJA G, et al.When more is better:multigene engineering in plants[J].Trends Plant Sci, 2010, 15(1):48-56. doi: 10.1016/j.tplants.2009.09.010
[17]	凌键, 陈永文, 龚一富, 等.甘薯离体遗传转化体系的优化[J].西南师范大学学报(自然科学版), 2004, 29(3):466-470. doi: 10.3969/j.issn.1000-5471.2004.03.032 LING J, CHEN Y W, GONG Y F, et al.Optimization of system for in vitro genetic transformation in sweetpotato[J].Journal of Southwest China Normal University (Natural Science Edition), 2004, 29(3):466-470. (in Chinese) doi: 10.3969/j.issn.1000-5471.2004.03.032
[18]	陈明利, 刘香利, 唐广立, 等.农杆菌侵染小麦的优化方案[J].分子植物育种, 2007, 5(4):577-582. doi: 10.3969/j.issn.1672-416X.2007.04.022 CHEN M L, LIU X L, TANG G L, et al.Optimization of Agrobacterium infecting Wheat[J].Molecular plant breeding, 2007, 5(4):577-582. (in Chinese) doi: 10.3969/j.issn.1672-416X.2007.04.022
[19]	奚亚军, 张启发, 林拥军, 等.利用农杆菌浸种法将叶片衰老抑制基因PSAG12-IPT导入普通小麦的研究[J].中国农业科学, 2004, 37(8):1235-1238. doi: 10.3321/j.issn:0578-1752.2004.08.027 XI Y J, ZHANG Q F, LIN Y J, et al.Introduction of Leaf Aging Inhibitory Gene PSAG12-IPT into Common Wheat by Agrobacterium Soaking[J].Chinese Agricultural Science, 2004, 37(8):1235-1238. (in Chinese) doi: 10.3321/j.issn:0578-1752.2004.08.027
[20]	雷江丽, 王丹, 吴燕民, 等.农杆菌浸种法介导中华结缕草遗传转化体系的建立[J].农业生物技术学报, 2009, 17(5):865-871. doi: 10.3969/j.issn.1674-7968.2009.05.021 LEI J L, WANG D, WU Y M, et al.Agrobacterium tumefaciens seed soaking method mediates the establishment of genetic transformation system of Zoysia japonica[J].Journal of Agricultural Biotechnology, 2009, 17(5):865-871. (in Chinese) doi: 10.3969/j.issn.1674-7968.2009.05.021
[21]	田文忠.提高籼稻愈伤组织再生频率的研究[J].遗传学报, 1994, 3(5):27-31. http://d.old.wanfangdata.com.cn/Periodical/zgnxtb201133001 TIAN W Z.Study on the Improve of Regeneration Frequency of Mature Embryo-derived indica Rice Callus[J].J Genet Genom, 1994, 3(5):27-31. (in Chinese) http://d.old.wanfangdata.com.cn/Periodical/zgnxtb201133001
[22]	CHENG M, HU T, JEAMME L, et al.Desiccation of plant tissue post-Agrobacterium infection enhances T-DNA delivery and in creases stable transformation efficiency in wheat[J].In Vitro Cell Dev Biol Plant, 2003, 39:595-604. doi: 10.1079/IVP2003471
[23]	马艳丽, 叶兴国, 刘艳鹏, 等.农杆菌敏感小麦基因型的筛选研究[J].麦类作物学报, 2005, 25(6):6-10. doi: 10.3969/j.issn.1009-1041.2005.06.002 MA Y L, YE X G, LIU Y P, et al. Screening of wheat genotype sensitive to Agrobacterium tumefaciens infection[J].Journal of Triticeae Crops, 2005, 25(6):6-10. (in Chinese) doi: 10.3969/j.issn.1009-1041.2005.06.002
[24]	王文治, 蔡文伟, 冯翠莲, 等.高效快速甘蔗转基因方法探索[J].热带作物学报, 2012, 33(9):1619-1624. doi: 10.3969/j.issn.1000-2561.2012.09.017 WANG W Z, CAI W W, FENG C L, et al.Exploring the method of high efficient and rapid sugarcane transgenic[J].Journal of Tropical Crops, 2012, 33(9):1619-1624. (in Chinese) doi: 10.3969/j.issn.1000-2561.2012.09.017
[25]	项友斌, 梁竹青, 高明尉, 等.农杆菌介导的苏云金杆菌抗虫因cryIA(b)和cryIA(c)在水稻中的遗传转化及蛋白表达[J].生物工程学报, 1999, 15(4):494-500. doi: 10.3321/j.issn:1000-3061.1999.04.015 XIANG Y B, LIANG Z Q, GAO M W, et al.Agrobacterium mediated Transformation of Insecticidal BacillusthuringiensiscryIA (b) and cryIA(c) Genes and Their Expression in Rice[J].Chinese Journal of Biotechnology, 1999, 15(4):494-500. (in Chinese) doi: 10.3321/j.issn:1000-3061.1999.04.015