ABSTRACT
Modern people generally suffer from α-linolenic acid (ALA) deficiency, since most staple food oils are low in ALA content. Thus, the enhancement of ALA in staple oil crops is of importance. In this study, the FAD2 and FAD3 coding regions from the ALA-king species Perilla frutescens were fused using a newly designed double linker LP4-2A, driven by a seed-specific promoter PNAP, and engineered into a rapeseed elite cultivar ZS10 with canola quality background. The mean ALA content in the seed oil of PNAP:PfFAD2-PfFAD3 (N23) T5 lines was 3.34-fold that of the control (32.08 vs 9.59%), with the best line being up to 37.47%. There are no significant side effects of the engineered constructs on the background traits including oil content. In fatty acid biosynthesis pathways, the expression levels of structural genes as well as regulatory genes were significantly upregulated in N23 lines. On the other hand, the expression levels of genes encoding the positive regulators of flavonoid-proanthocyanidin biosynthesis but negative regulators of oil accumulation were significantly downregulated. Surprisingly, the ALA level in PfFAD2-PfFAD3 transgenic rapeseed lines driven by the constitutive promoter PD35S was not increased or even showed a slight decrease due to the lower level of foreign gene expression and downregulation of the endogenous orthologous genes BnFAD2 and BnFAD3.
Subject(s)
Brassica napus , Brassica rapa , Perilla , Humans , Brassica napus/genetics , Brassica napus/metabolism , alpha-Linolenic Acid/chemistry , Fatty Acid Desaturases/genetics , Fatty Acid Desaturases/metabolism , Brassica rapa/genetics , Brassica rapa/metabolism , Seeds/genetics , Seeds/metabolism , Oils/metabolismABSTRACT
BACKGROUND: Hematopoietic stem cells (HSCs) have the ability to differentiate into all subsets of blood cells and self-renew. Large tumor suppressor 1 (LATS1) and large tumor suppressor 2 (LATS2) kinases are essential for cell cycle regulation, organism fitness, genome integrity, and cancer prevention. Here, we investigated whether Lats1 and Lats2 are critical for the maintenance of the self-renewal and quiescence capacities of HSCs in mice. METHODS: Quantitative reverse transcription-polymerase chain reaction was used to determine the expression levels of Lats1 and Lats2 in subsets of progenitor cells and mature bone marrow cells. A clustered regularly interspaced short palindromic repeats system was used to generate Lats1 or Lats2 knockout mice. Complete blood cell counts were used to compare the absolute number of white blood cells, lymphocytes, monocytes, neutrophils, and platelets between Lats1 or Lats2 heterozygotes and littermates. Flow cytometry was used to assess the size of hematopoietic progenitor cells (HPCs) and HSC pools in Lats1 or Lats2 heterozygotes and littermates. The comparison between the two groups was analyzed using Student's t test. RESULTS: Lats1 and Lats2 were widely expressed in hematopoietic cells with higher expression levels in primitive hematopoietic cells than in mature cells. Lats1 or Lats2 knockout mice were generated, with the homozygotes showing embryonic lethality. The size of the HPC and HSC pools in Lats1 (HPC: wild-type [WT] vs. heterozygote, 220,426.77â±â54,384.796 vs. 221,149.4â±â42,688.29, Pâ=â0.988; HSC: WT vs. heterozygote, 2498.932â±â347.856 vs. 3249.763â±â370.412, Pâ=â0.105) or Lats2 (HPC: WT vs. heterozygote, 425,540.52â±â99,721.86 vs. 467,127.8â±â89,574.48, Pâ=â0.527; HSC: WT vs. heterozygote, 4760.545â±â1518.01 vs. 5327.437â±â873.297, Pâ=â0.502) heterozygotes were not impaired. Moreover, the depletion of Lats1 or Lats2 did not affect the overall survival of the heterozygotes (Lats1: Pâ=â0.654; Lats2: Pâ=â0.152). CONCLUSION: These results indicate that a single allele of Lats1 or Lats2 may be sufficient for normal hematopoiesis.
