Application of rice OsNRT2.1 gene in increasing content of manganese element in rice grains
A rice grain, osnrt2.1 technology, applied in the field of agricultural genetic engineering, to achieve the effect of improving absorption and accumulation
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Embodiment 1
[0021] 1. Cloning of rice gene OsNRT2.1 sequence
[0022] 1) Extraction of total RNA
[0023] Rice (Nipponbare) seedlings grow to the 3-leaf stage, treated with 0.2mM nitrate nitrogen for 6 hours, and immediately take the roots and put them in liquid nitrogen for frozen storage. Weigh about 0.1g of roots, grind them with liquid nitrogen, and add them fully To a 1.5ml centrifuge tube, quickly add 1ml Trizol reagent (purchased from Invitrogen, USA), shake well, and extract total RNA.
[0024] 2) Cloning of the full length of the OsNRT2.1 gene
[0025] The rice NRT2.1 gene series OsNRT2.1 (AB008519) was retrieved from the gene database of the NCBI website (http: / / www.ncbi.nlm.nih.gov / ). Primers were designed (below) to fish out the full-length sequence of OsNRT2.1 from a tissue cDNA library.
[0026] P1:5'-CCAAGGTACCATGGACTCGTCGACGGTGGGC-3'
[0027] P2:5'-GGCCACTAGTTTAGGCGTGCTCCGGCGAG-3'
[0028] Using the total RNA obtained in step 1) as a template, after reverse transcript...
Embodiment 2
[0037] The absorption of manganese element under the different nitrogen treatment of embodiment 2
[0038] First, we cultured rice wild-type lines (WT) in 0.5mM NH 4 + (0.5A), 2.5mM NH 4 + (2.5A), 0.5mM NO 3 - (0.5N) and 2.5mM N0 3 - (2.5N) in the nutrient solution for nitrogen. 0.5mM NO 3 - Can promote rice root growth and be better than rice roots under other conditions (see figure 2 ). The total nitrogen concentration in shoots and roots was significantly higher under normal nitrogen treatment than in rice under 0.5mM treatment, and with 2.5mM NH 4 + Roots and shoots accumulated more total nitrogen under the treatment conditions. Different from the total nitrogen concentration, at 0.5mM NO 3 -Under the same conditions, the root absorbs more manganese instead, at 2.5mM NH 4 + The accumulation of manganese in shoots under the condition was significantly lower than that of rice under other nitrogen treatments. From the perspective of rice as a whole, the acc...
Embodiment 3
[0039] Example 3 Expression patterns of OsNRT2s and OsNAR2.1 in the roots of transgenic lines
[0040] Since OsNRT2.1 is a high-affinity nitrate transporter, we cultured three transgenic lines (OE1 / 2 / 3) and wild-type (WT) rice overexpressing OsNRT2.1 in 0.5 mM NH 4 + / NO 3 - In the nutrient solution as a nitrogen source, we found that the expression of OsNRT2.1 in the root of the overexpression line was up-regulated by 4.5 times and 5.7 times compared with the wild type, respectively, and was not affected by the form of nitrogen. And there was no significant difference in the expression of OsNRT2.3a and OsNRT2.4 in the root of the overexpression line compared with the wild type. However, the expression of OsNAR2.1 in overexpressed roots was only up-regulated by 80% under the condition of nitrate treatment ( image 3 ).
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