Construction and Verification of an Eucalyptus Gene Editing Vector with Visual Selection Markers

doi:10.7525/j.issn.1673-5102.2021.05.020

Abstract

Abstract:

As one of the three fast-growing tree species in the world， Eucalyptus has high value in economic， ecological， medicinal and other aspects. Due to the high genetic heterozygosity of Eucalyptus， many major economic traits might be jointly regulated by multiple genes， and conventional gene editing methods could not meet the requirements for efficient screening of Eucalyptus target gene editing and transformation. Using mCherry fluorescent protein as a selection marker might reduce the identification workload after transformation. In this study， a CRISPR/Cas9 vector containing the 35S promoter to promote the mCherry marker gene was constructed using Eucalyptus urophylla as the material to perform efficient visual editing of the Eucalyptus gene. The mCherry fluorescent protein was used as the selection marker to screen positively transformed progenies， and the adventitious bud genome containing fluorescent markers were extracted for PCR identification and analysis. The results showed that the editing vector PHEE401-35S-mCherry was successfully constructed. After the callus of Eucalyptus urophylla transformed， there was obvious red fluorescence under the light of 580 nm， and the target fragment with the same size as 35S-mCherry band was amplified by PCR identification. The study provided a visual screening method for the Eucalyptus gene editing.

Key words: Eucalyptus, gene editing, mCherry, CRISPR/Cas9

CLC Number:

Q782

Ze-Chen WANG, Ya-Mei LIU, Le-Jun OUYANG, Li-Mei LI, Chu-Yan LIANG, Jing-Yin PAN. Construction and Verification of an Eucalyptus Gene Editing Vector with Visual Selection Markers[J]. Bulletin of Botanical Research, 2021, 41(5): 816-823.

Figures/Tables 9

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References 28

1	Ouyang L J，Li L M. Effects of an inducible aiiA gene on disease resistance in Eucalyptus urophylla × Eucalyptus grandis［J］.Transgenic Research，2016，25（4）：441-452.
2	朱育锋，肖智华，彭晚霞，等.广西不同龄级桉树人工林植物多样性和群落结构动态变化特征［J］.中南林业科技大学学报，2018，38（12）：38-44.
	Zhu Y F，Xiao Z H，Peng X X，et al.Dynamics of plant diversity and community structure of Eucalyptus plantation at different ages class in Guangxi［J］.Journal of Central South University of Forestry & Technology，2018，38（12）：38-44.
3	王志超，杜阿朋，陈少雄.我国桉树人工林现状及可持续经营对策研究［J］.桉树科技，2012，29（4）：58-62.
	Wang Z C，Du A P，Chen S X.Current situation and sustainable management strategy of eucalypt plantations in China［J］.Eucalypt Science & Technology，2012，29（4）：58-62.
4	欧阳乐军，曾富华.桉树分子育种研究进展［J］.分子植物育种，2008，6（6）：1146-1152.
	Ouyang L J，Zeng F H.Advances in molecular breeding of Eucalyptus［J］.Molecular Plant Breeding，2008，6（6）：1146-1152.
5	欧阳乐军，沙月娥，黄真池，等.尾巨桉不同类型愈伤组织内源激素含量差异比较［J］.中南林业科技大学学报，2014，34（12）：71-77，86.
	Ouyang L J，Sha Y E，Huang Z C，et al.Study on difference of callus of Eucalyptus urophylla × Eucalyptus grandis［J］.Journal of Central South University of Forestry & Technology，2014，34（12）：71-77，86.
6	Gaj T，Gersbach C A，Barbas C F.ZFN，TALEN，and CRISPR/Cas-based methods for genome engineering［J］.Trends in Biotechnology，2013，31（7）：397-405.
7	Barrangou R.Cas9 targeting and the CRISPR revolution［J］.Science，2014，344（6185）：707-708.
8	沙月娥，欧阳乐军，彭舒，等.桉树胚状体再生与遗传转化的研究进展［J］.植物生理学报，2012，48（4）：325-332.
	Sha Y E，Ouyang L J，Peng S，et al.Research progress in plantlet regeneration of somatic embryos and genetic transformation in Eucalyptus［J］.Plant Physiology Communications，2012，48（4）：325-332.
9	范春节，曾炳山，裘珍飞，等.桉树转基因研究进展［J］.浙江林业科技，2008，28（2）：65-72.
	Fan C J，Zeng B S，Qiu Z F，et al.Progress on genetic transformation of Eucalyptus［J］.Journal of Zhejiang Forestry Science and Technology，2008，28（2）：65-72.
10	Chen Z Z，Tsay J Y，Chung J D.Callus culture of Eucalyptus grandis × E.urophylla and preliminary studies on organogenesis and Agrobacterium transformation［J］.Taiwan Forestry Science，1996，11（1）：43-52.
11	González E R，De Andrade A，Bertolo A L，et al.Production of transgenic Eucalyptusgrandis × E.urophylla using the sonication-assisted Agrobacterium transformation（SAAT） system［J］.Functional Plant Biology，2002，29（1）：97-102.
12	Tournier V，Grat S，Marque C，et al.An efficient procedure to stably introduce genes into an economically important pulp tree（Eucalyptus grandis × Eucalyptus urophylla）［J］.Transgenic Research，2003，12（4）：403-411.
13	Ho C K，Chang S H，Tsay J Y，et al.Agrobacterium tumefaciens-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants［J］.Plant Cell Reports，1998，17（9）：675-680.
14	Jefferson R A，Kavanagh T A，Bevan M W.GUS fusions：beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants［J］.The EMBO Journal，1987，6（13）：3901-3907.
15	吴学龙，何海燕，刘智宏，等.eGfp/Gus融合基因植物表达载体的构建和转化验证［J］.浙江农业学报，2011，23（4）：645-650.
	Wu X L，He H Y，Liu Z H，et al.Construction and verification of a plant vector carrying an eGfp/Gus fusion reporter gene［J］.Acta Agriculturae Zhejiangensis，2011，23（4）：645-650.
16	薛丽香，童坦君，张宗玉.报告基因的选择及其研究趋向［J］.生理科学进展，2002，33（4）：364-366.
	Xue L X，Tong T J，Zhang Z Y.The choice and research trend of reporter gene［J］.Progress in Physiological Sciences，2002，33（4）：364-366.
17	李亚丽，刘中来，周洁，等.转mCherry基因水稻的遗传分析及T-DNA整合位点的研究［J］.分子植物育种，2012，10（2）：121-130.
	Li Y L，Liu Z L，Zhou J，et al.Genetic analysis of mCherry transgenic rice and molecular characterization of T-DNA integration sites in the rice genome［J］.Molecular Plant Breeding，2012，10（2）：121-130.
18	李莉梅，张婷婷，欧阳乐军，等.利用pHDE-Cas9构建巨桉CTX基因家族CRISPR载体［J］.南方农业学报，2018，49（3）：411-417.
	Li L M，Zhang T T，Ouyang L J，et al.Construction of CRISPR vector of Eucalyptus grandis CTX gene family by pHDE-Cas9［J］.Journal of Southern Agriculture，2018，49（3）：411-417.
19	吴乃虎.基因工程原理［M］.北京：科学出版社，1997.
	Wu N H.Principle of genetic engineering［M］.Beijing：Science Press，1997.
20	王关林，方宏筠.植物基因工程原理与技术［M］.北京：科学出版社，1998.
	Wang G L，Fang H Y.Principles and techniques of plant genetic engineering［M］.Beijing：Science Press，1998.
21	王笑春，李峰，蒋湘宁.桉树组织培养与再生系统建立研究［J］.河北建筑科技学院学报，2006，23（2）：71-74.
	Wang X C，Li F，Jiang X N.The establishment of tissue culture regeneration system of Eucalyptus［J］.Journal of Hebei Institute of Architectural Science and Technology，2006，23（2）：71-74.
22	Chowdhury S R，Hoque S，Akter N.Optimization of regeneration and Agrobacterium tumefaciens-mediated transient transformation systems for Australian native extremophile，Tripogon loliiformis［J］.Journal of King Saud University-Science，2020，32（8）：3476-3485.
23	谢耀坚.桉树组织培养研究进展［J］.世界林业研究，2000，13（6）：14-19.
	Xie Y J.Advances in tissue culture of Eucalyptus［J］.World Forestry Research，2000，13（6）：14-19.
24	于颖，刘佳欣，周美琪，等.用于植物基因研究的mCherry表达载体构建及定位表达［J］.植物研究，2017，37（2）：194-199.
	Yu Y，Liu J X，Zhou M Q，et al.Construction and expression of mCherry expression vector used in plant［J］.Bulletin of Botanical Research，2017，37（2）：194-199.
25	刘树雷，孙琳，吴广延，等.腺相关病毒AAV2/8-CaMKⅡα-ChR2-mCherry的组织学及通道功能鉴定［J］.第三军医大学学报，2015，37（13）：1297-1301.
	Liu S L，Sun L，Wu G Y，et al.Histological features and channel functions of adeno-associated virus AAV2/8-CaMKⅡα-ChR2-mCherry in glutamatergic neurons［J］.Journal of Third Military Medical University，2015，37（13）：1297-1301.
26	欧阳乐军，马铭赛，陈梓洛，等.一种拟南芥基因高效编辑体系研究［J］.植物研究，2019，39（6）：869-875.
	Ouyang L J，Ma M S，Chen Z L，et al.An efficient gene editing research system of Arabidopsis［J］.Bulletin of Botanical Research，2019，39（6）：869-875.
27	周丹.大豆二酰甘油酰基转移酶（DGAT）基因克隆及其功能的初步研究［D］南京：南京农业大学，2012.
	Zhou D.Cloning and primary characterization genes of diacylglycerol acyltransferase（DGAT） in soybea（Glycine max）［D］.Nanjing：Nanjing Agricultural College，2012.
28	马铭赛，布梁灏，陈自银，等.基于Cas9-Free的拟南芥ALB3基因编辑载体构建及验证［J］.华北农学报，2020，35（1）：44-50.
	Ma M S，Bu L H，Chen Z Y，et al.Construction and validation of ArabidopsisALB3 gene editing vector based on Cas9-Free［J］.Acta Agriculturae Boreali-Sinica，2020，35（1）：44-50.

引物名称 Primer name	引物序列（5′→3′） Primer sequence	引物用途 Primer use
35s-F	taatgcattttatgacttgcAACATGGTGGAGCACGACAC	载体构建 Vector construction
35s-R	CCAAGAGCTCCGTGTCCTCTCCAAATGAAA	载体构建 Vector construction
EMch-F	AGAGGACACGgagctcttggactcccatgt	载体构建 Vector construction
EMch-R	GAATTCGTTGTCAATCAATTAATACGATAATTTATTTGAA	载体构建 Vector construction
35s-GT1	ACCTCCTCGGATTCCATT	鉴定引物 Identification primer
EMch-GT2	AGATAAAGCCACGCACAT	鉴定引物 Identification primer

温度 Temperature	步骤 Step	时间 Time
94℃	预变性Initialization	4 min
94℃	变性Denaturation	30 s
56℃	退火Annealing	30 s
72℃	延伸Elongation	30s
72℃	最终延伸Final elongation	2 min
4℃	保存Save	∞

温度 Temperature	步骤 Step	时间 Time
94℃	预变性Initialization	4 min
94℃	变性Denaturation	30 s
56℃	退火Annealing	30 s
72℃	延伸Elongation	45 s
72℃	最终延伸Final elongation	4 min
4℃	保存Save	∞

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