| 王 越，姚晓华，吴昆仑，白羿雄，魏晓星.青稞 HVA1和 blt4.9基因对模拟水分胁迫的响应差异及其在抗旱育种中的应用[J].麦类作物学报,2019,(6):666 |
| 青稞 HVA1和 blt4.9基因对模拟水分胁迫的响应差异及其在抗旱育种中的应用 |
| Difference of HVA1 and blt4.9 Gene Expression Patterns under Simulated Drought Stress and the Potention Application in Drought Tolerance Breeding in Hulless Barley |
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| DOI：10.7606/j.issn.1009-1041.2019.06.06 |
| 中文关键词: 青稞 HVA1基因 blt4.9基因 内参基因 实时荧光定量PCR |
| 英文关键词:Hullessbarley HVA1 blt4.9 Reference genes Real-time quantitative PCR |
| 基金项目:国家自然科学基金项目(31660388；31160284)；现代农业产业技术体系建设专项(CARS-05)；青海省科技厅项目（2019-ZJ-7075;2017-NK-A7） |
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| 中文摘要: |
| 为了解青稞 HVA1和 blt4.9基因在抗旱方面的作用及其差异，以青稞品种昆仑12号为材料，利用RT-PCR从8个候选内参基因（ DHN1、 GAPDH、 Actin-1、 Actin-2、 18SrRNA-1、 18SrRNA-2、 TC139057和 PKABA）中筛选出在15%PEG模拟水分胁迫下表达稳定的基因，同时以筛选到的稳定表达的基因为参照，采用qRT-PCR技术研究青稞 HVA1基因和 blt4.9基因在模拟水分胁迫条件下的表达差异及在不同抗旱性青稞品种鉴定中的应用。结果表明：(1)筛选到1个在干旱胁迫下青稞叶片中能稳定表达的内参基因 TC139057 ；(2)在1%～30%PEG模拟干旱胁迫下，随着胁迫时间的延长，青稞 HVA1和 blt4.9基因表达量呈先增后降趋势，25%PEG处理下表达量最高，且在较低浓度PEG处理下基因表达量为 HVA1＞ blt4.9，而在较高浓度PEG处理下反之；(3)15%PEG处理1～144 h，两基因表达量也呈先增后降趋势，胁迫48 h后 HVA1基因表达量最高，胁迫96 h后 blt4.9基因表达量最高，且处理前期基因表达量为 HVA1＞ blt4.9，后期反之；(4) 1～500 μmol·L-1 ABA处理下，两基因表达量仍呈先增后降趋势， HVA1基因比 blt4.9基因敏感，且在较低ABA浓度下基因表达量为 HVA1＞ blt4.9，在较高浓度下反之；(5)获得转青稞 HVA1和 blt4.9基因拟南芥植株，其主要抗旱性生理指标优于野生型；且在较轻胁迫下，转 HVA1植株的生理指标优于转 blt4.9植株，而在较重胁迫处理下情况相反；(6)在模拟水分胁迫下，青稞叶片中两基因的表达量，抗旱性强的显著高于抗旱性弱的材料(P＜0.01)，且较轻胁迫下 HVA1基因较敏感，反之 blt4.9基因敏感。本研究结果为 HVA1和 blt4.9基因在青稞抗旱育种中的应用奠定了基础。 |
| 英文摘要: |
| In order to study the difference roles of drought resistance genes HVA1 and blt4.9 in hulless barley under drought, eight candidate internal reference genes (DHN1, GAPDH, actin-1, actin-2, 18SrRNA-1, 18SrRNA-2, TC139057 and PKABA) were screened in Kunlun 12 under 15% PEG. The stable reference gene was used to study the differences of HVA1 and blt4.9 gene expression in hulless barley under simulated drought stress and their application in the identification of different drought-resistance cultivars. The results showed that:a stable reference gene TC139057 was selected undersimulated drought stress in hulless barley; using TC139057 as the reference gene, the expression levels of HVA1 and blt4.9 genes in hulless barley were higher than those of the control (P<0.01) under the simulated drought stress, which showed a change trend of first increasing and then decreasing. The highest expression levels of these two genes appeared under 25% PEG treatment. Under lower concentration of PEG treatment (1%-20%), the expression level of HVA1 was higher than that of blt4.9, but under the higher concentration of PEG (25%-30%), the expression level of blt4.9 was higher. The two genes stably expressed from 1 to 144 h under 15% PEG treatment, and the expression level increased first and then decreased with the prolonging of stress time. The peak of expression level for HVA1 and blt4.9 appeared at 48 h and 96 h, respectively. The gene expression level of HVA1 was higher than that of blt4.9 from 1 h to 48 h, which is in contrast to that from 96 h to 144 h.Under the ABA treatment (1-500 μmol·L-1), the expression levels of the two genes also showed a change trend of first increasing and then decreasing.The highest expression level of HVA1 gene appeared under 10 μmol·L-1 ABA, but that of blt4.9 gene appeared under 50 μmol·L-1 ABA, which was significantly different from the control (P<0.01). The expression level of HVA1 was higher than that of blt4.9 under 1-10 mmol·L-1 ABA, in contrast to that under 50-500 mmol·L-1 ABA. The transgenic HVA1 and blt4.9 Arabidopsis plants were achieved. The main drought-resistance physiological indices of transgenic plants of hulless barley were greater than those of wild-type. The protein content in transgenic HVA1 plant was highest under 10% PEG stress, followed by that intransgenic blt4.9 plant and wild type WT. However, under 20% and 30% PEG stress, the protein content in transgenic blt4.9 plant was highest, following by that in transgenic HVA1 plant and wild type WT. Therelative conductivity and malondialdehyde content were opposite to proteins. Under the simulated drought stress, the expression of the two genes in the leaf of the droughtresistant hulless barley was higher than that of the susceptible ones (P<0.01). The HVA1 gene expressed abundantly at early stage of drought stress or under moderate drought stress while the blt4.9 gene expressed abundantly at late stage of drought stress or under severe drought stress. Therefore, we can use the expression characteristics of these two genes in hulless barley breeding for drought resistance. |
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