Overexpression of Pinus massoniana PmPT3 Gene in Arabidopsis thaliana Increasing Low Phosphorus Tolerance

doi:10.13560/j.cnki.biotech.bull.1985.2021-0072

Abstract

Abstract:

The aim of this study is to explore the effect of PmPT3 gene of Pinus massoniana on phosphorus uptake and utilization in Arabidopsis thaliana under low-phosphorus stress,and to achieve the utilization of excellent genes in trees. The PmPT3 gene overexpression vector pBWA（V）HS-PmPT3 was constructed and transformed into A. thaliana by inflorescence infection method. Four homozygous strains of T3 generation were obtained by screening and detection. The results of phosphorus treatment showed that overexpression of the PmPT3 gene of P. massoniana significantly increased the activities of superoxide dismutase（SOD）,peroxidase（POD）and catalase（CAT）in transgenic Arabidopsis,respectively,it was 2.17 times,1.59 times,and 1.81 times of the wild-type plant. It reduced malondialdehyde（MDA）content,47.58% lower than that of wild-type plants. Compared with the wild-type,the total phosphorus content and inorganic phosphorus content of the aerial parts and roots of transgenic Arabidopsis increased by 1.26 times and 1.74 times,as well as 1.38 times and 1.89 times,respectively. Compared with the wild type,the dry weight of the transgenic plant shoots increased by 45.46%,the dry weight of roots by 55.56%,and the total dry weight by 46.15%.Compared with normal phosphorus supply conditions,the expressions of PmPT3 gene in the root and shoot parts of transgenic A. thaliana under low phosphorus stress was significantly up-regulated,and reached a very significant level in the roots. These results indicated that the overexpression of PmPT3 gene from P. massoniana increased the activities of protective enzymes,reduced the production of MDA,and promoted the absorption of phosphorus in Arabidopsis under low phosphorus stress. Therefore,overexpression of the PmPT3 gene improved the ability of Arabidopsis to tolerate low phosphorus stress. This work may provide a reliable scientific basis for the creation of new low phosphorus tolerant germplasm through genetic engineering.

Key words: overexpression, Pinus massoniana, PmPT3 gene, Arabidopsis thaliana, low phosphorus stress

FANG Dan-dan, ZHANG Ting, WEN Xiao-peng. Overexpression of Pinus massoniana PmPT3 Gene in Arabidopsis thaliana Increasing Low Phosphorus Tolerance[J]. Biotechnology Bulletin, 2021, 37(10): 1-8.

Figures/Tables 8

Table 1 Primers and sequences used in the study

引物Primer	序列Sequence（5'-3'）	退火温度Annealing temperature/℃
PmPT3-F	CAGTGGTCTCACAACATGGGC- GACAATGAGGGG	55
PmPT3-R	CAGTGGTCTCATACACTACAC- GGGCATAGTTCTGT	55
Actin2-qRTF	ACGGTAACATTGTGCTCAGT- GGTG	59
Actin2-qRTR	CTTGGAGATCCACATCTGCTGGA	59
PmPT3-qRTF	CTATCCGCTTTCAGCCACCA	59
PmPT3-qRTR	TTCTCCACACAAAATCCGCC	59

Fig.1 Construction of plant expression vector of P. masso-niana PmPT3 Gene M: DL2000 DNA marker; A: PmPT3 fragment with adding restriction sites; B: BLUNT-PmPT3 enzyme digestion product; C: pBWA(V)HS denzyme digestion product; D: recombinant plasmid pBWA(V) HS-PmPT3 enzyme digestion product; E: 1-11 colony PCR product

Fig.2 Process of genetic transformation of P. massoniana PmPT3 into Arabidopsis LP: Low phosphorus (0.125 mmol/L) ; TG; transgenic plants; A, B: T1 generation transgenic Arabidopsis selection; C, D: T2 generation transgenic Arabidopsis selection; E, F: screening of T3 generation pure commonzanara; G, H: phenotype of Arabidopsis thaliana after 20 days of phosphorus treatment

Fig.3 Analysis of positive gene expression in T1 generation transgenic Arabidopsis thaliana (A) and PCR detection of homozygous strains (B) Different lowercase letters in the figure indicate significant differences at the P<0.05 level

Fig.4 Effect of low phosphorus stress on the POD(A), SOD(B), CAT(C) activity and MDA(D) content of transgenic Arabidopsis LP: Low phosphorus (0.125 mmol/L); TG: transgenic plants; different lowercase letters in the figure indicate significant differences at the P<0.05 level. The same below

Fig.5 Effect of low phosphorus stress on the total phosphorus content (A) and inorganic phosphorus content (B) of transgenic Arabidopsis

Fig.6 Effect of low phosphorus stress on the above-ground dry weight (A), root dry weight (B), total dry weight (C) and root to shoot ratio (D) of transgenic Arabidopsis

Fig.7 Relative expression of PmPT3 gene of P. massoni-ana after phosphorus treatment

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