Genetic transformation and resistance evaluation of glyphosate resistance gene in Brassica napus

doi:10.19802/j.issn.1007-9084.2021259

Abstract

Abstract:

A novel glyphosate-resistant gene （designated as I. variabilis-EPSPS?） was cloned from bacterium of Isoptericola variabilis. In this research, I.variabilis-EPSPS* was overexpressed in Brassica napus L. cv Yu 127, under the control of constitutive CaMV35S promoter. Seeds from I.variabilis-EPSPS* transgenic plants could germinate and grow well in the presence of increasing concentrations of glyphosate（40-80 mg L^-1）. In contrast, wild type line had significantly reduced shoot and root length. qRT-PCR indicated that expression level of I.variabilis-EPSPS* increased after isopropylamine salt （Roundup） treatment, and while expression remained unchanged. After spraying 600× dilution of 41% glyphosate Roundup （1/3 recommended dose in field）, the cumulative amount of shikimic acid in I.variabilis-EPSPS* transgenic T₁ plants was lower than wild type. Additionally, agronomic performance demonstrated no significant difference in silique length, seeds per silique, and 1000-seed weight （etc.） between I.variabilis-EPSPS* E₁T₁ and wild type in greenhouse. Foliar glyphosate application in field showed that glyphosate at concentration of 759.8 mg L^-1 had no detrimental effect on the growth of I.variabilis-EPSPS* plants. Furthermore, the glyphosate-sprayed transgenic plants were preriodically monitored until maturity, and it was observed that they exhibited normal growth and morphological characteristics similar to those of WT which was not spayed with glyphosate in the greenhouse. In summary, the I.variabilis-EPSPS* gene and its transgenic glyphosate-resistant B. napus L. line E₁ could be the new materials for genetical modified rapeseed breeding resistant to glyphosate herbicide.

Key words: I.variabilis-EPSPS*genes, Brassica napus, transgene, herbicide resistance

CLC Number:

S565.4

Shuai DU, Li-li WAN, Zhuan-rong WANG, Yi XU, Deng-feng HONG, Guang-sheng YANG. Genetic transformation and resistance evaluation of glyphosate resistance gene in Brassica napus[J]. CHINESE JOURNAL OF OIL CROP SCIENCES, 2022, 44(6): 1199-1209.

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References 34

1	赵波. 甘蓝型油菜矮秆基因定位、克隆及功能分析［D］. 武汉：华中农业大学， 2017.
2	苏少泉. 稻田杂草对除草剂的抗性及其防治［J］. 农药， 2001， 40（7）： 11-14. DOI：10.16820/j.cnki.1006-0413.2001.07.002 . doi: 10.16820/j.cnki.1006-0413.2001.07.002
3	Green J M. Evolution of glyphosate-resistant crop technology［J］. Weed Sci， 2009， 57（1）： 108-117. DOI：10.1614/ws-08-030.1 . doi: 10.1614/ws-08-030.1
4	Duke S O， Powles S B. Glyphosate： a once-in-a-century herbicide［J］. Pest Manag Sci， 2008， 64（4）： 319-325. DOI：10.1002/ps.1518 . doi: 10.1002/ps.1518
5	Cerdeira A L， Duke S O. The current status and environmental impacts of glyphosate-resistant crops［J］. J Environ Qual， 2006， 35（5）： 1633-1658. DOI：10.2134/jeq2005.0378 . doi: 10.2134/jeq2005.0378
6	Dill G M. Glyphosate-resistant crops： history， status and future［J］. Pest Manag Sci， 2005， 61（3）： 219-224. DOI：10.1002/ps.1008 . doi: 10.1002/ps.1008
7	Roberts F， Roberts C W， Johnson J J， et al. Evidence for the shikimate pathway in apicomplexan parasites［J］. Nature， 1998， 393（6687）： 801-805. DOI：10.1038/31723 . doi: 10.1038/31723
8	Dill G M， Cajacob C A， Padgette S R. Glyphosate-resistant crops： adoption， use and future considerations［J］. Pest Manag Sci， 2008， 64（4）： 326-331. DOI：10.1002/ps.1501 . doi: 10.1002/ps.1501
9	苏少泉. 草甘膦述评［J］. 农药， 2005， 44（4）： 145-149. DOI：10.16820/j.cnki.1006-0413.2005.04.001 . doi: 10.16820/j.cnki.1006-0413.2005.04.001
10	Bradshaw L D， Padgette S R， Kimball S L， et al. Perspectives on glyphosate resistance［J］. Weed Technol， 1997， 11（1）： 189-198. DOI：10.1017/s0890037x00041567 . doi: 10.1017/s0890037x00041567
11	Shaner D L. Lessons learned from the history of herbicide resistance［J］. Weed Sci， 2014， 62（2）： 427-431. DOI：10.1614/ws-d-13-00109.1 . doi: 10.1614/ws-d-13-00109.1
12	Shaner D L， Lindenmeyer R B， Ostlie M H. What have the mechanisms of resistance to glyphosate taught us？［J］. Pest Manag Sci， 2012， 68（1）： 3-9. DOI：10.1002/ps.2261 . doi: 10.1002/ps.2261
13	Pollegioni L， Schonbrunn E， Siehl D. Molecular basis of glyphosate resistance-different approaches through protein engineering［J］. Febs J， 2011， 278（16）： 2753-2766. DOI：10.1111/j.1742-4658.2011.08214.x . doi: 10.1111/j.1742-4658.2011.08214.x
14	Wakelin A M， Preston C. A target-site mutation is present in a glyphosate-resistant Lolium rigidum population［J］. Weed Res， 2006， 46（5）： 432-440. DOI：10.1111/j.1365-3180.2006.00527.x . doi: 10.1111/j.1365-3180.2006.00527.x
15	Preston C， Wakelin A M. Resistance to glyphosate from altered herbicide translocation patterns［J］. Pest Manag Sci， 2008， 64（4）： 372-376. DOI：10.1002/ps.1489 . doi: 10.1002/ps.1489
16	Vila-Aiub M M， Balbi M C， Distéfano A J， et al. Glyphosate resistance in perennial Sorghum halepense （Johnsongrass）， endowed by reduced glyphosate translocation and leaf uptake［J］. Pest Manag Sci， 2012， 68（3）： 430-436. DOI：10.1002/ps.2286 . doi: 10.1002/ps.2286
17	柳寒，周永明. 抗草甘膦基因mEPSPS转化油菜研究［J］. 中国油料作物学报， 2012， 34（6）： 582-585.
18	王景雪，赵福永，徐培林，等. 油菜转抗草甘膦、抗虫基因获得双抗植株［J］. 遗传学报， 2005， 32： 1293-1300.
19	万丽丽，王转茸，辛强，等. GOX-CP4EPSP转化甘蓝型油菜的抗草甘膦研究［J］. 湖北农业科学， 2016， 55（10）： 2661-2666. DOI：10.14088/j.cnki.issn0439-8114.2016.10.053 . doi: 10.14088/j.cnki.issn0439-8114.2016.10.053
20	Comai L， Sen L C， Stalker D M. An altered AroA gene product confers resistance to the herbicide glyphosate［J］. Science， 1983， 221（4608）： 370-371. DOI：10.1126/science.221.4608.370 . doi: 10.1126/science.221.4608.370
21	Yang X， Li L， Jiang X Q， et al. Genetically engineered rice endogenous 5-enolpyruvoylshikimate-3-phosphate synthase （epsps） transgene alters phenology and fitness of crop-wild hybrid offspring［J］. Sci Rep， 2017， 7： 6834. DOI：10.1038/s41598-017-07089-9 . doi: 10.1038/s41598-017-07089-9
22	Green J M. The rise and future of glyphosate and glyphosate-resistant crops［J］. Pest Manag Sci， 2018， 74（5）： 1035-1039. DOI：10.1002/ps.4462 . doi: 10.1002/ps.4462
23	Zhou M， Xu H L， Wei X L， et al. Identification of a glyphosate-resistant mutant of rice 5-enolpyruvylshikimate 3-phosphate synthase using a directed evolution strategy［J］. Plant Physiol， 2006， 140（1）： 184-195. DOI：10.1104/pp.105.068577 . doi: 10.1104/pp.105.068577
24	李杰华. 抗广谱性除草剂转基因油菜创制及抗性评价［D］. 武汉：华中农业大学， 2018.
25	Yi S Y， Wu G B， Lin Y J， et al. Characterization of a new type of glyphosate-tolerant 5-enolpyruvyl shikimate-3-phosphate synthase from Isoptericola variabilis ［J］. J Mol Catal B： Enzym， 2015， 111： 1-8. DOI：10.1016/j.molcatb.2014.11.009 . doi: 10.1016/j.molcatb.2014.11.009
26	Cui Y， Huang S Q， Liu Z D， et al. Development of novel glyphosate-tolerant Japonica rice lines： a step toward commercial release［J］. Front Plant Sci， 2016， 7： 1218. DOI：10.3389/fpls.2016.01218 . doi: 10.3389/fpls.2016.01218
27	Cui Y， Li C Y， Zhou F， et al. Developing of transgenic glyphosate-tolerant Indica restorer line with commercial application potential［J］. Mol Breed， 2020， 40（4）： 1-13. DOI：10.1007/s11032-020-01124-w . doi: 10.1007/s11032-020-01124-w
28	李杰华，端群，史明涛，等. 新型抗广谱性除草剂草甘膦转基因油菜的创制及其鉴定［J］. 作物学报， 2021， 47： 778-787.
29	Zhou Y， Wang H， Gilmer S， et al. Control of petal and pollen development by the plant cyclin-dependent kinase inhibitor ICK1 in transgenic Brassica plants［J］. Planta， 2002， 215（2）： 248-257. DOI：10.1007/s00425-002-0752-2 . doi: 10.1007/s00425-002-0752-2
30	Song X L， Wu J J， Zhang H J， et al. Occurrence of glyphosate-resistant horseweed （Conyza canadensis） population in China［J］. Agric Sci China， 2011， 10（7）： 1049-1055. DOI：10.1016/S1671-2927（11）60093-X . doi: 10.1016/S1671-2927（11）60093-X
31	娄远来，邓渊钰，沈晋良，等. 甲磺隆和草甘膦对空心莲子草乙酰乳酸合酶活性和莽草酸含量的影响［J］. 植物保护学报， 2005， 32（2）： 185-188. DOI：10.3321/j.issn： 0577-7518.2005.02.015 . doi: 10.3321/j.issn： 0577-7518.2005.02.015
32	Liu X E， Li X G， Hai L， et al. How efficient is film fully-mulched ridge-furrow cropping to conserve rainfall in soil at a rainfed site？［J］. Field Crops Res， 2014， 169： 107-115. DOI：10.1016/j.fcr.2014.09.014 . doi: 10.1016/j.fcr.2014.09.014
33	Watson A， Ghosh S， Williams M J， et al. Speed breeding is a powerful tool to accelerate crop research and breeding［J］. Nat Plants， 2018， 4（1）： 23-29. DOI：10.1038/s41477-017-0083-8 . doi: 10.1038/s41477-017-0083-8
34	Wang Z， Wan L， Zhang X， et al. Interaction between Brassica napus polygalacturonase inhibition proteins and Sclerotinia sclerotiorum polygalacturonase： implications for rapeseed resistance to fungal infection［J］. Planta， 2021， 253（2）： 34. DOI：10.1007/s00425-020-03556-2 . doi: 10.1007/s00425-020-03556-2

引物名称 Primer name	序列（5’-3’） Sequence （5’-3’）	用途 Usage	扩增产物 Amplification （gene）
P1	ATTAGCGCTAGGGACGTGAG	PCR	I.variabilis-EPSPS*
P2	ATACGCTCCCACATCCTGTC	PCR	I.variabilis-EPSPS*
P3	GGAGGGTCTTCGCAGTCG）	qRT-PCR	I.variabilis-EPSPS*
P4	GCTGTGCTCGGTCTTAGGG	qRT-PCR	I.variabilis-EPSPS*
P5	GGAAGCTCCTGGAATCCATGAGA	qRT-PCR	Actin
P6	TCTTTGCTCATACGGTCAGCAATTCC	qRT-PCR	Actin

培养基种类 Medium type	培养基组成 Medium composition	pH
发芽培养基 Germination medium	MS＋20 g/L Sucrose （蔗糖）＋0.8% Agar （琼脂）	5.8~5.9
悬浮培养基 Suspension medium	MS＋200 μmol AS（乙酰丁香酮）	5.4~5.5
预培养培养基 Pre-culture medium	MS＋30 g/L Sucrose＋1 mg/L 6-BA＋1 mg/L 2，4-D＋8.0 g/L Agar	5.8~5.9
共培养培养基 Co-culture medium	MS＋30 g/L Sucrose＋18 g/L Mannitol （甘露醇）＋1 mg/L 2，4-D＋0.3 mg/L KT （激动素， kinetin）＋100 μmol/L AS＋8.0 g/L Agar	5.8~5.9
脱菌培养基 Sterilization medium	MS＋30 g/L Sucrose＋18 g/L Mannitol＋1 mg/L 2，4-D＋0.3 mg/L KT＋100 μmol/L AS＋8.0 g/L Agar	5.8~5.9
芽再生培养基 Germination medium	MS＋10 g/LGlucose （葡萄糖）＋0.25 g/L Xylopyranose （木糖）＋0.6 g/L MES （吗啉乙磺酸）＋2.0 mg/L ZT＋0.1 mg/L IAA＋300 mg/L Timentin （抑菌剂）＋60 mg/L Glyphosate（草甘膦）＋8 g/L Agar	5.8~5.9
生根培养基 Root regeneration medium	MS＋10 g/L Sucrose＋300 mg/L Timentin＋8.0 g/L Agar	5.8~5.9

HIS	草甘膦处理下植株的受损症状Injury symptoms on glyphosater-treated plants
0	植株生长正常No symptom
1	植株生长受到轻微抑制，极少叶片掉落，没有其他受损症状 Plant growth inhibited slightly. Entire plant wilted， all leaves droop slightly， but no other symptom occurred
2	包括嫩叶在内少于30%的叶片卷曲和坏死 Entire plant wilts including the young leaves. Leaf apex curls and etiolates. Less than 30% of leaves have necrotic
3	幼叶卷曲和坏死，整株植物30%~60%的叶片枯萎 Young leaves curls and etiolate. Entire plant etiolates. 30-60% of leaves are dead
4	整株植物叶片60%~80%枯萎死亡 Young leaf curls more. Entire plant etiolates more. 60-80% of leaves are dead
5	植株超过80%的叶片死亡或整个植株死亡 Entire plant wilts completely. More than 80% of leaves are dead， or entire plant dead

农达稀释倍数 Dilution multiples of Roundup	损伤率Damage rate /%
农达稀释倍数 Dilution multiples of Roundup	E₁T₁	育127 Yu 127
0	0	0
800×	0	60
400×	40	68
200×	52	80

材料名称Material （稀释倍数Dilution factor）	表型 Phenotype			考察数量 Test number
材料名称Material （稀释倍数Dilution factor）	角果长 SL /mm	每角果粒数 NSS /mm	千粒重 TSW /g	考察数量 Test number
E₁T₁（0）	55.40±1.02a	25.40±1.62a	2.85±0.04a	10
E₁T₁（1000×）	54.20±2.99a	23.80±2.79a	2.80±0.13a	10
E₁T₁（800×）	54.40±2.07a	25.20±2.28a	2.79±0.07a	10
E₁T₁（600×）	53.60±1.02a	23.60±1.02a	2.74±0.20a	10
育127 Yu 127 （0）	54.80±2.39a	24.40±3.36a	2.83±0.06a	10
育127 Yu 127 （1000×）	—	—	—	10
育127 Yu 127 （800×）	—	—	—	10
育127 Yu 127 （600×）	—	—	—	10