Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (24): 4982-4991.doi: 10.3864/j.issn.0578-1752.2020.24.002

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Acquisition and Characteristic Analysis of Transgenic Maize with phyA2, ZmTMT, and Bar

YAO XingLan(),YANG WenZhu,LUO YanZhong,CHEN RuMei,WANG Lei(),ZHANG Lan()   

  1. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081
  • Received:2020-03-07 Accepted:2020-04-20 Online:2020-12-16 Published:2020-12-28
  • Contact: Lei WANG,Lan ZHANG E-mail:15751004149@163.com;wanglei01@caas.cn;zhanglan01@caas.cn

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Abstract:

【Objective】Corn seeds are the main feed ingredient for poultry and monogastric animals, but the content of α-tocopherol with high-activity in corn seeds is low, and maize is rich in phosphorus mainly existed in the form of phytate, which cannot be effectively utilized by animals. Therefore, synthesized DL-α-tocopheryl acetate and phosphorus or microbial phytase are supplemented in the feed for optimal animal growth. These methods not only greatly increase the feed cost, but also cause the phosphorus pollution due to the undigested phytate. The aim of this study is to acquire maize seeds rich in α-tocopherol and high in phytase activity, and provide resources for forage maize breeding.【Method】Construct an expression vector with three expression cassette, and introduced into maize through agrobacterium infection method, glufosinate was used for screening; The positive transgenic plants were identified by spraying glufosinate and PCR analysis; RT-PCR analysis of target gene expression at the transcription level; Western blot analysis of target gene expression at the translation level; Phytase activity was measured by spectrophotometric method; vitamin E content was determined by HPLC. At the same time, the differences of other nutrients and agronomic traits between transgenic lines and wild types were compared.【Result】Constructed an expression vector with three expression cassette, phyA2 driven by endosperm-specific promoter 123387, ZmTMT driven by embryo-specific promoter 13387, Bar driven by constitutive promoter CaMV35S, and introduced into maize; Transgenic plants were identified by spraying glufosinate and PCR; Two transgenic homozygous maize lines TPB1 and TPB2 with good target traits were obtained after multiple generations of backcross transfer. RT-PCR and Western blot analysis showed that phyA2, ZmTMT and Bar were all highly expressed in transgenic lines. Phytase activity determination showed phytase activity reached to 10 000-13 000 U·kg -1, which is able to meet the needs of animal growth and development, vitamin E content measurement showed more than 90% of γ-tocopherol was transformed to α-tocopherol, and α-tocopherol content increased to 50-70 mg·kg -1. In addition, there were no significant differences on agronomic traits between transgenic lines and wild type, and there was no significant difference on nutrient component between TPB1 and wild type, slight difference but without adverse effect between TPB2 and wild type; and the transgenic plants were resistant to BASTA.【Conclusion】These transgenic maize, which are rich in α-tocopherol and phytase, and also with herbicide resistance, can be applied for maize breeding to reduce feed cost, improve the utilization rate of phosphorus and reduce environmental pollution.

Key words: maize, phytase, vitamin E, α-tocopherol, glufosinate resistance

Fig. 1

Schematic diagram of vector construction phyA2 cassette is driven by endosperm-specific promoter 123387; ZmTMT cassette is driven by embryo-specific promoter 13387; Bar cassette is driven by the constitutive promoter CaMV 35S"

Fig. 2

RT-PCR analysis of phyA2, ZmTMT and Bar genes in maize seeds 15 days after pollination WT: Wild type Zheng58; TPB1, TPB2: Transgenic homozygous lines"

Fig. 3

Western blot analysis of phyA2, ZmTMT and Bar in mature maize seeds A, B, and C show the protein accumulation of ZmTMT, Bar and phyA2, respectively; WT: Wild type Zheng58; TPB1, TPB2: Transgenic homozygous lines; ZmTMT CK: Positive control of ZmTMT; Bar CK: Positive control of Bar; phyA2 CK: Positive control of phyA2"

Fig. 4

Analysis of phytase activity in maize seeds WT: Wild type Zheng58; TPB1, TPB2: Transgenic homozygous lines"

Table 1

Analysis of the contents in phytic acid and phosphorus of maize seeds"

株系
Lines		 植酸磷
Phytic acid (mg·g-1)		 磷
Phosphorus (mg·g-1)
WT 2.77±0.41 0.22±0.02
TPB1 2.60±0.22 0.22±0.06
TPB2 2.65±0.19 0.20±0.02

Fig. 5

Analysis of vitamin E content in maize seeds A: Chromatogram of vitamin E determination. α-, γ-, δ-T/T3: α-, γ-, δ-tocopherol/tocotrienol, IS Internal standard: Rac-5,7-dimethyltocol; B: Analysis of Vitamin E content in maize seeds. WT: Wild type Zheng58; TPB1, TPB2: Transgenic homozygous lines"

Table 2

Analysis of other nutrient components in maize seeds (%)"

营养成分Nutrient component WT TPB1 TPB2
天门冬氨酸 Aspartic acid 0.55±0.001 0.54±0.004 0.58±0.005
苏氨酸 Threonine 0.33±0.001 0.32±0.001 0.36±0.004
丝氨酸Serine 0.41±0.003 0.40±0.002 0.46±0.003*
谷氨酸 Glutamic acid 1.46±0.008 1.42±0.006 1.72±0.001**
脯氨酸 Proline 0.88±0.009 0.8±0.007* 0.97±0.015**
甘氨酸 Glycine 0.32±0.005 0.31±0.002 0.33±0.002
丙氨酸 Alanine 0.59±0.001 0.57±0.002 0.68±0.005**
胱氨酸 Cystine 0.18±0.001 0.19±0.003 0.21±0.007
缬氨酸 Valine 0.42±0.010 0.41±0.002 0.47±0.002*
蛋氨酸 Methionine 0.31±0001 0.44±0.005** 0.38±0.005*
异亮氨酸 Isoleucine 0.28±0.001 0.27±0.001 0.32±0.001*
亮氨酸 Leucine 1.02±0.075 0.99±0.004 1.22±0.007**
酪氨酸 Tyrosine 0.30±0.001 0.24±0.008* 0.25±0.005*
苯丙氨酸 Phenylalanine 0.43±0.003 0.40±0.010 0.47±0.010*
组氨酸 Histidine 0.23±0.001 0.23±0.007 0.26±0.008*
赖氨酸 Lysine 0.26±0.001 0.26±0.005 0.28±0.008
精氨酸 Arginine 0.37±0.002 0.36±0.010 0.38±0.002
色氨酸 Tryptophan 0.07±0.001 0.07±0.001 0.07±0.001
钙 Calcium 0.03±0.001 0.03±0.001 0.03±0.001
粗蛋白 Crude protein 8.47±0.063 8.35±0.008 9.89±0.050**
粗脂肪 Crude fat 3.10±0.015 2.50±0.001* 2.80±0.010
干物质 Dry matter 91.10±1.330 91.80±1.410 90.80±1.270

Fig. 6

Analysis of agronomic traits WT: Wild type Zheng58; TPB1, TPB2: Transgenic homozygous lines"

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