| Tobacco NtFerl is an important gene involved in the ferritin synthesis, ferrtitin plays important in iron ions storage and stress reaction. In order to test and verity the function of tobacco NtFerl, T1 generation of NtFerl overexpression transgenic tobaccos were taken as the materials to analysis their physiological characteristics. The results showed that in different Fe2+ levels, the relative expression of exogenous NtFerl more stable, while the relative expression of endogenous NtFerl higher than the amount of the blade root, and Fe2+ concentration increased with increasing. NtFerl overexpresstion could increase the growth, SOD and POD activity, total iron content in leaves and roots while decreased the MDA content, and it also could increase the chlorophyll relative content while inhibit NtFerl transferred Fv/Fm decreased speed.Heilongjiang Province is the main producing area of japonica in China, the genetic improvement of japonica quality using genetic engineering breeding has an important economic value and social significance. The NtFerl transgenic japonica were got basic on the establishment and optimization of Songjing 6, Kongyu 131 and Dongnong 427 tissue culture system. The main conclusions were as the follows:1. The callus induction rate of Songjing 6 and Kongyu 131 were 88.9% and 81.3% under 2mg/L 2,4-D withlmg/L 6-BA concentration respectively, the callus induction rate of Dongnong 427 was 54.3% under 3mg/L 2,4-D with lmg/L 6BA concentration and Embryo was rather strong. Adding lmg/L 6-BA into callus culture medium was good for increasing the japonica callus induction rate. NMB+2mg/L 6-BA+lmg/L KT+0.5mg/L NAA was the differentiation medium for Songjing6 and Kongyu131 while NMB+2mg/L 6-BA+0.5mg/L KT+0.5mg/L NAA was the differentiation medium for Dongnong427.2. By Agrobacterium-mediated method, we transfered the NtFerl into Songjing6, Kongyu131 and Dongnong427 and got the transgenic lines. The PCR、Southern blot and Northern blot test showed the gene had been integrated into the japonica’s genome and expressed. After that, we got the seeds of transgenic lines. The results of T1 and T2 generation kanamycin resistance screening, PCR and RT-PCR test showed exogenous gene NtFerl had been stability genetic and expressed in T1 and T2 generation. The relative expression had no significant difference in different Fe2+ levels, periods and tissue parts.3. Comparison analysis of SOD, POD activity and MDA, chlorophyll content between transgenic lines and CK showed:NtFerl could increase antioxidant enzyme activity or delay the activity decline rate, decrease MDA content, thus it could reduce oxidative damage to cells and increase the chlorophyll content of transgenic plants.4. The total iron content test analysis in root, leaves and brown rice under different Fe2+ concentration showed:NtFerl could increase the total iron content in root, leaves and brown rice. The total iron content of 3 transgenic varities brown rice were 5.98%-24.36% more than the CK, among all transgenic lines, Nt3 of Songjing6, Ntl of Kongyul31 and Nt3 of Dongnong 427 were the best ones.5. Results of Fe-Zn correlation in roots, leaves and brown rice showed:NtFerl could decrease antagonistic level in leaves and brown rice under 300μM Fe2+ concentration, thus increase the total iron content in leaves and brown rice while decrease the inhibition of Zn absorption and unilization leaded by the iron overload.6. The leaf blast resistance result of transgenic lines showed:NtFerl could increase the resistance to leaf blast, especially under 300μM Fe2+ concentration, the leaf blast index of Songjing6, Kongyu 131 and Dongnong 427 were 6.7%,11.1% and 8.6% less than the CK respectively.7. The free amino acids of these 3 transgenic varieties were all increased under different Fe2+ concentration, but the total amino acids content of Songjing 6 and Dongnong 427 were less than the CK, Glutamic acid, alanine and leucine were more than the CK. NtFerl could also provide changes of agronomic traits, such as the tillers were increased while seed setting rate and 1000-grain weight were decrease significant.8. Nt3 of Songjing6, Ntl of Kongyu 131 and Nt3 of Dongnong 427 were selection as the T2 transgenic generation. The physiological characteristics analysis of T2 showed:the SOD activity of transgenic plants were 1.4%-14.7% more than CK, MDA content were 2.3%-14.8% less than the CK, root length (Diameter≥ 0.45mm) was 3.2%-30.6% more than the CK, the total iron content in leaves and brown rice were 2.0%-18.9% and 7.9%-21.2% more than the CK respectively. The maximal fluorescence was also increase in transgenic lines, the chlorophyll content, optimal/maximal photochemical efficiency of PS Ⅱ in the dark (Fv/Fm), photochemical efficiency of PS Ⅱ in the light (Fv’/Fm’) and photochemical quenching (qP) were all increased under 5μM Fe2+ (Iron deficiency) and 300μM Fe2+ (Iron overload) concentration. The leaf blast index was 6.2%-12.1% less than the CK under 300μM Fe2+ concentration, it indicated that NtFer 1 could increase the blast resistance of japonica. |
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