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Functional Analysis of Two Rice Nitrate Transporter Genes OsNRT1.1a and OsNRT1.1b

Author: ZuoQiSong
Tutor: XuGuoHua;FanXiaoRong
School: Nanjing Agricultural College
Course: Plant Nutrition
Keywords: Rice N Nitrate transporter Gene expression Transgenic rice Electrophysiology
CLC: S511
Type: Master's thesis
Year: 2011
Downloads: 10
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China’s agriculture has entered the erea of high-cost times. The seeds, ferilizers, pesticides and other prices have been generally increased and the rural labor force is also greatly reduced. China’s agriculture has entered the times of the quality of agricutural products and environmental protection. Nitrogen fertilizer plays a crucial role in the rice production. In recent years, Chinese uses of nitrogen fertilizer is also sharply increased. Rice planting area in China accounts for 20% of the total area of the world, but they account for the world’s nitrogen fertilizer application rate of 37% in total. In 1995 China’s nitrogen fertilizer production and use have reached first in the world, but China’s nitrogen fertilizer use efficiency is low, also aggravated the pollution of the environment, leading to ecological deterioration. Therefore, by biological means to increase the utilization of nitrogen fertilizer in rice, reduce nitrogen pollution, protect natural resources, agricultural production is currently a relatively good way. Plant uptake of nitrate is absorbed through the root system by transport protein system to complete nitrate transporter.There are high-affinity and low affinity uptake system to complete the transportion. Through the rice nitrate transporter gene cloning, we got two highly homologous rice nitrate transporter gene OsNRTl.1a and OsNRT1.1b. In this study, model variety Nipponbare (Nipponbare) were used, the function and expression regulation paterns were studied by RT-PCR, electrophysiological and overexpresstion. The main results obtained are as follows:1. OsNRT1.1a and OsNRT1.1b obtained 95% homology by anaysised of gene, the amino acid sequence and ransmembrane. Both have only one intron difference. But the ORF longthe of OsNRT1.1a is two times of OsNRT1.1b,and the ORF of OsNRT1.1b is the first part of OsNRT1.1a.OsNRT1.1b is 6 times transmembrane by transmembrane prediction analysis. Sequence analysis showed that OsNRT1.1a and OsNRT1.1b both located on chromosome 3,with the same promoter and a single copy in the rice genome.2. Analyzes the gene expression of OsNRT1.1a and OsNRT1.1b in rice shoots and underground by RT-PCR technology, The results showed that they are not subject to different nitrogen forms and concentrations. This two genes has some similarities on the expression by different treatments. They don’t vary with different nirogen forms and different concentrations.This two genes are relatively weak under full nitrogen. when a low concenration of NO3- or NH4+ induced,the corresponding increased in the expression.So these two genes maybe constitutively expressed induced by nitrigen..3.OsNRT1.1a and OsNRT1.1b were coloned and overexpressed mediaed by agrobacterium through rice callus. These two transgenic plants were treated by different forms and concentrations,and the results showed that these two transgenic plants are significantly higher than WT plants and taller,more tillers and seed set higher.and with the same result treated in soil culture conditions.we analysis the total N of underground and aboveground parts,and the total N of two transgenic plants of aboveground is higher than WT but underground of the total nitrogen content is not fixed.the transgenic plants and wt were cultured at the same time,and the transgenic plants were significantly higher than wild-type after a week culture.The Length of shoot and root was recored every four days.The results showed that the both transgenic plants grow faster than WT,and final average of about 5 cm higher than WT.but the underground part of transgenic plants and WT did not differ significantly.4. Electrophysiology analyse showed that after overexpresstion of OsNRT1.1a and OsNRT1.1b in rice,the Overexpression of OsNRT1.1a (OEa) has no singinficent difference to WT treated with High and low concentrations of nitrate. The overexpression of OsNRT1.1b(OEb) on low concentration of nitrate is only a weak response but with no response on high concentration of nitrate. the response of OEa was similar to WT treated with high and low concentrations of ammonium, but OEb has a strong response on high and low concentrations of annonium.Low concentrations of nitrate induced expression OEa basic and similar to wild-type. High concentrations of nitrate in response to strong conditions, reflect the lower affinity of OsNRT1.1a nitrate transporter gene characteristics.5. Heterologous expression in Xenopus showed that,when inject OsNRT1.1b cRNA,it depolarizated about 10mv to low concentrations of NO3-,So Xenopus may absorb certain NO3-.So OsNRT1.1b may be responsible for a nitrate transporter gene.

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