Genetic transformation and growth index determination of the Larix olgensis LoHDZ2 transcription factor gene in tobacco

Abstract

Homeodomain-leucine zippers (HD-Zip) are plant-specific transcription factors that participate in different plant development processes and differentially regulate metabolic processes. LoHDZ2 is an HD-ZipII subfamily transcription factor gene that we identified from a transcriptomic analysis of Larix olgensis. To understand its function, we built a LoHDZ2 expression vector and then inserted it into tobacco by genetic transformation. Transgenic plants were identified at the DNA and RNA levels. Phenotypic index analysis of transgenic tobacco showed dwarfed growth with larger leaves and earlier flowering than the wild type. LoHDZ2 was expressed differently after hormone treatment with IAA, MeJA and 2,4-D. The results suggested that LoHDZ2 may respond to hormones and be involved in regulating growth and metabolism. These results helped us better understand the function of LoHDZ2 and provided a candidate gene for Larix olgensis molecular breeding.

Figure 1

Diagram of the pCAMBIA1302 overexpression vector.

Figure 2

LoHDZ2 expression levels under different hormone treatments. *Compared with 0 h treatment, the difference was significant; **Compared with 0 h treatment, the difference was extremely significant.

Figure 3

Double restriction digestion of the target gene and vector. M: 2000 bp DNA molecular marker; 1–8: transgenic lines.

Figure 4

PCR detection of the target gene in Agrobacterium. M: 2000 bp DNA molecular marker; 1–12: transgenic lines.

Figure 5

LoHDZ2 subcellular positioning results. (A) D dark field observation; (B) E tom observation; (C) F is the combination of both.

Figure 6

Growth status of wild-type tobacco in different concentrations of hyg.

Figure 7

Resistance screening of genetically modified tobacco. (a) Cocultivation; (b) Induced resistance of differentiated buds; (c) Rooted tissue culture seedlings; (d) Transplantation into soil.

Figure 8

PCR detection of p1302-LoHDZ2 in transgenic tobacco. M: 2000 bp DNA molecular marker; 1: negative control; 2: water; 3: positive control; 4–10: transgenic strains.

Figure 9

Relative expression levels of p1302-LoHDZ2 in transgenic tobacco. WT: wild tobacco; OE2-OE5: different transgenic lines.

Figure 10

Changes in the plant height of transgenic tobacco grown for 30 days, 60 days and 90 days, as well as changes in leaf size after 90 days of growth. (a) Wild-type and transgenic tobacco grown for 30 days; (b) Wild-type and transgenic tobacco grown for 60 days; (c) Wild-type and transgenic tobacco grown for 90 days; (d) Wild-type and transgenic tobacco leaves grown for 90 days. WT: wild-type tobacco; OE2-OE5: different transgenic lines.

Figure 11

Phenotypic observation of transgenic tobacco during the reproductive period. (a), (c) Transgenic and wild-type tobacco floral organs; (b) Transgenic and wild-type tobacco fruits; (d) Unopened transgenic and wild-type tobacco flowers; (e) Transgenic and wild-type tobacco plants. WT: wild-type tobacco; OE2-OE5: different transgenic lines.

Figure 12

Determination of the growth index of transgenic tobacco and wild-type tobacco. (a) Plant height of transgenic tobacco and wild-type tobacco; (b) High growth rate of transgenic tobacco and wild-type tobacco; (c) Leaf length of transgenic tobacco and wild-type tobacco; (d) Leaf length change rate in transgenic tobacco and wild-type tobacco; (e) Leaf width of transgenic tobacco and wild-type tobacco; (e) Leaf width change rate of transgenic tobacco and wild-type tobacco; WT: wild tobacco; OE2-OE5: different transgenic lines.

Figure 13

Relative expression levels of transgenic and wild-type tobacco under different hormone treatments. (a), (b) Tobacco treated with 2,4-D hormone; (c) ,(d) Tobacco treated with MeJA hormone; (e), (f) Tobacco treated with IAA hormone; WT wild tobacco, OE2-OE5 different transgenic lines.