Molecular Cloning And Expression Of The NHX1 Promoters From Oryza Sativa And Chenopodium Glaucum

Agricultural productivity is severely affected by soil salinity because salt levels that are harmful to plant growth affect large terrestrial areas of the world. The damaging effects of salt accumulation in agricultural soils have influenced ancient and modern civilizations. It is estimated that 20% of the irrigated land in the world is presently affected by salinity. Two main approaches are being used to improve salt tolerance: (i) the exploitation of natural genetic variations, either through direct selection in stressful environments or through mapping quantitative trait loci and subsequent marker-assisted selection; and (ii) the generation of transgenic plants to introduce novel genes or to alter expression levels of the existing genes to affect the degree of salt stress tolerance. Here, we discuss the NHX gene which is a functional tool for the development of salt-tolerant crops. NHX1,a tonoplast Na+/H+ antiporter energized by the ?pH across the tonoplast,facilitates vacuolar compartmentalization of the cation. As a fundamental mechanism in salt tolerance,an active antiporter would function to sequester Na+ into the vacuole, which avoid of cytoplasmic Na+ toxicity and maintenance of a high cytoplasmic K+/Na+ ratio. In parallel, vacuolar Na+ would serve as an osmoticum necessary for cellular H2O homeostasis.Promoter is important to decide gene when and where expression. It plays the main role in the transgenic technic especially inducible promoter. Using inverted-PCR (I-PCR) method, a novel promoter of NHX1 (Vacuolar Na+/H+ exchanger) was cloned from halophyte species Chenopodium glaucum, CgNHX1. The sequence is -1~ -3800bp in the upstream of CgNHX1. The different length of promoters including 2000bp, 1000bp and 500bp were cloned by PCR from genomic DNA of Oryza sativa and Chenopodium glaucum. Then these promoters were constructed into pCAMBIA1391Z and the activity of promoters was detected by transient expression in onion epidermis. All different length of promoters had basal activity. The length is longer and the activity is stronger. The activity of promoters was analyzed under different stresses including salt, cold, ABA and drought. The activity of CgNHX1 and OsNHX1 promoters were enhanced by salt, drought and ABA treatment but not cold treatment. The activity of OsP2000, OsP1000 and OsP500 was increased by ABA treatment, however, only CgP2000 could be induced by ABA treatment but not CgP1000 and CgP500.Improvement of plants by genetic engineering open up new possibilities for phyto-remediation. Tissue culture is prerequisite to plant genetic engineering. Chenopodium glaucum may be useful in remediation of soil salinity by transgenic engineering. But there is no report on tissue culture of Chenopodium glaucum. So we have developed axenic Chenopodium glaucum cultures and studied the effects of phytohormone, sugar and pH on its growth.. And this is also helpful for the study of expression comparison of the NHX1 promoters from Oryza sativa and Chenopodium glaucum.First,the effects of explants, illumination and plant growth regulator on callus induction of Chenopodium glaucum were studied. The results showed that the best callus induction medium was MS medium with NAA4.0 mg/L+6-BA0.5 mg/L for the stem and MS with 2,4-D4.0 mg/L+6-BA0.2 mg/L for the leaf respectively. The calli were induced more efficiently by light treatment than dark. Then callus subculture was optimized, that is cultured with the addition of Vc 5.0mg/L,2,4-D0.5mg/L+6-BA0.5mg/L, and the callus could inhibit browning effectively with Vc after continuous subculture. The MS medium with 6-BA2.0 mg/L+NAA0.05 mg/L was preferable for inducing adventitious shoot .And the 1/2MS medium with NAA0.2 mg/L was suitable for the root differentiation.Second, a protocol for high frequency adventitious shoot induction and plantlet regeneration from immature inflorescence explants of Chenopodium glaucum was reported. Adventitious shoots were induced from immature inflorescence cultured on Murashige and Skoog (MS) medium supplemented with 1.0 mg/L 6-benzylaminopurine (6-BA) and 0.4 mg/L 3-indole butyric acid (IBA) for 35 days. Induction frequency of adventitious shoots was about 66.7%. Adventitious shoots were further multiplied vigorously and maintained for a long time on the same medium for adventitious shoot induction. Rooting was achieved on 1/2MS + NAA 0.2 mg/L medium after adventitious shoot was transplanted for 2-3 weeks. Third, a protocol for high frequency adventitious shoot induction and plantlet regeneration from stem explants of Chenopodium glaucum was established. Adventitious shoots were induced from stem on medium MS+6-BA 1.5 mg/L + IAA 0.4 mg/L; Rapid Propagation medium was MS+6-BA 1.0 mg/L +IAA 0.6 mg/L; Rooting was achieved on 1/2MS + NAA 0.2 mg/L medium.