Ilhma2, the heavy metal ATPase transporter of S.
A technology for transporting proteins and heavy metals, applied in the fields of molecular biology and genetic engineering, can solve unreported problems, improve transport capacity, enhance tobacco's tolerance and enrichment capacity for cadmium, and enhance cadmium tolerance Effects of Sexuality and Enrichment Ability
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Embodiment 1
[0027] The cloning of embodiment 1 Malus sinensis heavy metal ATPase transporter gene IlHMA2
[0028] Cultivate the water cultured to 8 weeks of horseshoe seedlings in 100mg / L CdCl 2 2.5H 2 Stress treatment in the Hoagland nutrient solution of O for 24 hours, cut the roots and wash them with sterile water, put them on sterile filter paper to absorb the water, weigh 100 mg of fresh roots, grind them fully in liquid nitrogen, and follow the TaKaRa MiniBEST Plant RNA Extraction Kit The total RNA was extracted according to the kit instructions, and the concentration was measured by a Quawell5000 nucleic acid protein analyzer, and the integrity of the RNA was detected by 1.2% formaldehyde denaturing gel electrophoresis. Take 1 μg of total RNA, and perform reverse transcription according to the instructions of the TaKaRaPrimeScriptRTase cDNA first-strand synthesis kit. , to synthesize cDNA.
[0029] According to the HMA nucleotide sequence of the plant heavy metal ATPase transporter...
Embodiment 2
[0043] Example 2 The predictive analysis of the structural characteristics of the heavy metal ATPase transporter IlHMA2
[0044] Using the TMpred program (http: / / www.ch.embnet.org / software / TMPRED_form.html) to predict the transmembrane region of IlHMA2 in S. chinensis, IlHMA2 has 8 transmembrane regions (TM1-TM8). The homology between IlHMA2 and the HMA2 proteins of wheat, rice and Arabidopsis plants was 68%, 66% and 55% respectively through the blast comparison of the NCBI website, and the function prediction of the IlHMA2 protein showed that it contained heavy metal P 1B -ATPase-specific GICCPSE, CPC, DKTGT, HP and GDGMNDAPAL motifs ( figure 1 ). Among them, the motif with metal binding domain contained in the N-terminus is GICCPSE, which belongs to the functional domain of metal-related regulation; the CPC site is responsible for metal ion conduction, and the DKTGT is the phosphorylation binding site for enzyme phosphorylation; the HP site Involved in the translocation of...
Embodiment 3
[0045] Phylogenetic tree analysis of embodiment 3 Malus sinensis heavy metal ATPase transporter gene IlHMA2
[0046] In order to analyze the genetic relationship between the IlHMA2 gene and other plant HMA genes, MEGA6.0 software was used to analyze other plants (Arabidopsis AhHMA2, Arabidopsis lyre AlHMA2, Arabidopsis AtHMA2 / 4, mustard BjHMA4, turnip BrHMA2, Rapeseed BnHMA2 / 4, Camelina CasHMA2 / 4, Clementine CcHMA2, Orange CisHMA2, Shepherd’s Purse CrHMA2, Cucumber CsHMA2 / 4, Behenia EsHMA2, Barley HvHMA2, IlHMA2 IlHMA2, Medicago truncatula MtHMA2, Celery sativa NcHMA4, short anther wild rice ObHMA2, rice OsHMA2, Populus euphratica PeHMA2, castor RcHMA2, sorghum SbHMA2, potato StHMA2, wheat TaHMA2, cocoa TcHMA3, grape VvHMA3, maize ZmHMA2) HMA genes were analyzed by phylogenetic tree. The results showed that IlHMA2 was closely related to monocotyledonous plants such as wheat TaHMA2 gene, rice OsHMA2 gene, and barley HvHMA2 gene, while its genetic relationship with dicotyledonou...
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