CN105779478A - 玉米ZmAGA4基因及其应用 - Google Patents

玉米ZmAGA4基因及其应用 Download PDF

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CN105779478A
CN105779478A CN201610207096.8A CN201610207096A CN105779478A CN 105779478 A CN105779478 A CN 105779478A CN 201610207096 A CN201610207096 A CN 201610207096A CN 105779478 A CN105779478 A CN 105779478A
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赵天永
张明帅
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Northwest A&F University
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Abstract

本发明涉及玉米ZmAGA4基因及其应用。发明人通过超表达玉米GRMZM2G077181基因(命名为ZmAGA4)从而提高植物抗热的方法。通过同源克隆，用PCR方法克隆了玉米ZmAGA4基因。构建了用35S启动子调控该基因的表达载体。将该载体用农杆菌介导方法转入植物拟南芥后，转基因植株的抗热能力显著提高。该基因超表达植株在正常条件下生长正常；在高温胁迫条件下该转基因植株生长优于对照，且能促进植物早熟，缩短生长周期。

Description

玉米ZmAGA4基因及其应用
技术领域
本发明属于生物科学技术领域，具体涉及玉米ZmAGA4基因及其提高植物抗高温能力的应用和促进植物早熟的应用。
背景技术
通过调控相关基因表达提高植物抗热的研究多有报道，但多数转基因植物由于在正常条件下植物生长受到抑制，因此到目前为止，仍未见相关基因应用于商业生产。
发明内容
玉米ZmAGA4基因属于一类α-半乳糖苷酶(α-Galactosidase，EC3.2.1.22)(图1)。发明人研究发现玉米ZmAGA4启动子－589bp位置有一个响应热激元件，在受到热激胁迫时能够诱导ZmAGA4基因的上调表达。在拟南芥中过表达玉米ZmAGA4基因，发现拟南芥的抗热性显著增强，热激后的成活率显著高于对照组，说明玉米ZmAGA4基因在玉米抗热过程中发挥着直接作用。
发明人通过PCR方法克隆了玉米GRMZM2G077181基因(命名为ZmAGA4)，将该基因在拟南芥中超表达，提高了植物抗热能力。该基因过表达植株在正常条件下生长正常，在高温胁迫条件下该基因生长优于对照，且能促进植物早熟，缩短生长周期。
为此，本发明目的之一是提供玉米ZmAGA4基因，其序列如SEQNO1所示。
本发明目的之二是提供玉米ZmAGA4基因提高植物高温性能的应用。
本发明的目的之三是提供玉米ZmAGA4基因促进植物生长的应用。
与现有技术相比，本发明的有益效果在于：
在植物中过量表达玉米ZmAGA4基因能够提高植物的抗热能力。过量表达玉米ZmAGA4基因对植物在正常环境条件下的生长没有副作用，且能促进植物早熟，缩短生长周期。
附图说明
图1.玉米ZmAGA4蛋白序列进化分析显示玉米ZmAGA4属于α-半乳糖苷酶家族；
图2.鉴定ZmAGA4基因启动子上有一个HSE元件；
图3.拟南芥转化载体pCsGFPBT基本框架示意图；
图4.ZmAGA4基因表达载体模式图及转基因植株鉴定(实时定量)；
图5.过表达ZmAGA4提高了拟南芥抗热性。(a)未经高温处理时各株系表型。WT为野生型，OXGFP为过表达GFP株系，OXAGA4-7，OXAGA4-9为过表达ZmAGA4转基因株系；(b)高温处理及生长条件示意图；(c)高温处理后植株表型；(d)各株系存活率统计，每个株系至少3个生物学重复；星号*表示差异显著(P<0.05)；
图6.过表达AGA4促使拟南芥早熟；(a)同一批次种子在相同生长时期的各拟南芥株系的表型；(b)相同生长时期的各拟南芥株系第一个侧枝；(c)-(g)分别表示相同生长时期的各实验拟南芥株系的主茎长度、侧枝数目、开花数目、果荚数目、第一个侧枝长度，以上统计有多个生物学重复，不同字母表示具有显著性差异。
具体实施方式
1、发明人通过PCR方法克隆了玉米ZmAGA4基因的编码区，构建了该基因植物表达载体。
对三叶期玉米幼苗进行42度热激处理。从处理8小时的叶片提取RNA并将其反转录成cDNA。
以cDNA为模板，用上游引物：
5’-cgggatccTGACGGTGACGCCGAGGATTAC-3’
和下游引物：
5’-gctctagaCTAGACTTCGATCTCCAGGCTCCATC-3’
对ZmAGA4的编码区进行PCR扩增。
扩增程序为：98℃预变性2min；98℃变性10s，60℃退火2.5min，35个循环；72℃终延伸8min。
将扩增产物切胶回收，用BamHI和XbaI酶切片段，后与经改造过的载体pCsGFPBT(用退火片段F：5’-CATGGCTCAACTCGG-3’和R：5’-GATCCCGAGTTGAGC-3’替换载体中的GFP基因，见下图3)连接。将ZmAGA4的ORF片段连在载体的BamHI和XbaI位点之间，构建超表达拟南芥载体。载体有植物转化筛选标记潮霉素基因(见图4a)。载体上的ZmAGA4ORF片段经测序正确后转入农杆菌AGL1，用花序侵染法侵染拟南芥Col0野生型。T0代种子经过70％乙醇室温1min，20％次氯酸钠室温5min消毒，用无菌水清洗7遍后，均匀涂布在MS平板上(含潮霉素25mg/L)。4℃处理3天后转移至22℃培养箱生长。萌发约7-10天，转基因植株的叶深绿，根较长；非转化的植株叶浅绿，根短，不能长期存活。将转化体移入营养土中生长直到收T1代种子。T1代种子按上述方法筛选，收T2代种子(每个株系收多个单株)。每个T2代单株种子分别筛选，种植，收集每个单株的T3代种子，筛选每个单株的T3代种子，后代不分离的(均对潮霉素有抗性)进行下一步鉴定。
2、发明人发现在植物中过表达玉米ZmAGA4基因提高了植物抗高温能力。
转基因拟南芥苗期抗热性检测：将MS培养基上生长(22℃，16/8光周期，相对湿度60％)7天的拟南芥幼苗移至45℃培养箱，高温处理100分钟。热激处理后放置于22℃条件下(16/8光周期，相对湿度60％)恢复培养14天后测定存活率。
在拟南芥中超表达玉米ZmGolS2基因提高了植物抗高温能力，参见图5。
3、发明人发现在植物中过表达玉米ZmAGA4基因促进了植物早熟。
同时收获的拟南芥新鲜种子，干燥后在无抗MS萌发，后同时移栽至土壤中，在22℃条件下(16/8光周期，相对湿度60％)正常生长约30天，拟南芥长势出现明显差异，即过表达ZmAGA4的两个拟南芥株系生长速度明显快于野生型和过表达GFP的拟南芥植株，而使拟南芥开花结果和提前，生长周期缩短，促进植物早熟。

Claims (3)

1.玉米ZmAGA4基因序列如SEQNO1所示。
2.玉米ZmAGA4基因提高植物高温性能的应用。
3.玉米ZmAGA4基因促进植物生长的应用。
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115948366A (zh) * 2022-11-16 2023-04-11 西北农林科技大学 玉米ZmAGA1基因用于提高植物抗旱性的应用
CN116254287A (zh) * 2022-11-24 2023-06-13 西北农林科技大学 玉米ZmAGA3基因用于提高植物耐旱性的应用
CN116254287B (zh) * 2022-11-24 2024-06-04 西北农林科技大学 玉米ZmAGA3基因用于提高植物耐旱性的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8962915B2 (en) * 2004-06-14 2015-02-24 Evogene Ltd. Isolated polypeptides, polynucleotides encoding same, transgenic plants expressing same and methods of using same
CN104946662A (zh) * 2015-04-21 2015-09-30 西北农林科技大学 玉米ZmGolS2基因提高植物抗旱、抗高温、抗盐能力的应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8962915B2 (en) * 2004-06-14 2015-02-24 Evogene Ltd. Isolated polypeptides, polynucleotides encoding same, transgenic plants expressing same and methods of using same
CN104946662A (zh) * 2015-04-21 2015-09-30 西北农林科技大学 玉米ZmGolS2基因提高植物抗旱、抗高温、抗盐能力的应用

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
AKSHAY KAKUMANU ET AL.: "Effects of Drought on Gene Expression in Maize Reproductive and Leaf Meristem Tissue Revealed by RNA-Seq", 《PLANT PHYSIOLOGY PREVIEW》 *
GENBANK: "PREDICTED: Zea mays probable galactinol--sucrose galactosyltransferase 2 (LOC103644222), mRNA，NCBI Reference Sequence: XM_008667421.3", 《GENBANK》 *
MEI-LIANG ZHOU ET AL.: "Genome-wide identification of genes involved in raffinose metabolism in Maize", 《GLYCOBIOLOGY》 *
TIAN-YONG ZHAO ET AL.: "An alkaline a-galactosidase transcript is present in maize seeds and cultured embryo cells, and accumulates during stress", 《SEED SCIENCE RESEARCH》 *
赵天永: "通过调控棉子糖代谢途径提高玉米种子活力和耐逆性", 《全国农业生化与分子生物学第群届学术研讨会》 *
韩庆辉: "ZmAGA1基因5’调控区的功能分析", 《中国优秀硕士学位论文全文数据库 基础科学辑》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115948366A (zh) * 2022-11-16 2023-04-11 西北农林科技大学 玉米ZmAGA1基因用于提高植物抗旱性的应用
CN115948366B (zh) * 2022-11-16 2024-04-09 西北农林科技大学 玉米ZmAGA1基因用于提高植物抗旱性的应用
CN116254287A (zh) * 2022-11-24 2023-06-13 西北农林科技大学 玉米ZmAGA3基因用于提高植物耐旱性的应用
CN116254287B (zh) * 2022-11-24 2024-06-04 西北农林科技大学 玉米ZmAGA3基因用于提高植物耐旱性的应用

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